Fibrosis biomarker assay

ABSTRACT

Provided herein are methods of diagnosis or of quantitation of fibrosis. An immunoassay is conducted to measure neo-epitope containing protein fragments of collagen type III, collagen type I, collagen type IV, collagen type V, or collagen type VI, elastin, biglycan, decorin, lumican, versican, perlecan, neurocan, brevican, fibromodulin, serglycin, syndecan, betaglycan, vimentin, or C-reactive protein naturally present in a biofluid sample obtained from a patient. An above normal elevation of the measured protein fragments in the patient is associated with the presence or extent of fibrosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/EP2008/064946 filed on Nov. 4, 2008, which claims Convention priority from GB0721713.6 filed in the United Kingdom on Nov. 5, 2007, GB0722748.1 filed in the United Kingdom on Nov. 20, 2007 and GB0802814.4 filed in the United Kingdom on Feb. 15, 2008, and also claims the benefit under 35 U.S.C. §1.119(e) of U.S. Provisional application No. 61/211,467 filed on Mar. 30, 2009 and U.S. Provisional application No. 61/289,081 filed on Dec. 22, 2009. The entire contents of each of the aforementioned patent applications are incorporated herein by this references.

INCORPORATION BY REFERENCE TO MATERIAL SUBMITTED ON A COMPACT DISC

A sequence listing file is submitted herewith.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to assays for biomarkers useful in the diagnosis of fibrosis disease and prognosis of its development, including biomarkers indicative of the risk of developing fibrosis after a chronic injury.

In particular, according to the present invention, biomarkers relating to degradation fragments of Collagen type I, III, IV, V, and VI, elastin, C-reactive protein, and proteoglycans including Biglycan, Decorin, Versican, and Perlecan are found to be useful.

2. Description of Related Art

Fibrotic diseases (including those listed in Table 1) are a leading cause of morbidity and mortality, e.g. cirrhosis with 800,000 death per year worldwide¹.

TABLE 1 Different fibrotic diseases² Tissue Examples of Causes Liver Viral hepatitis Schistosomiasis Steatohepatitis (Alcoholic or non-alcoholic) Lung Idiopathic pulmonary fibrosis (IPF) Systemic sclerosis (Scleroderma) Kidney Nephrogenic systemic fibrosis (NSF) Diabetes Untreated hypertension Heart Heart attack Hypertension Atherosclerosis Restenosis Eye Macular degeneration, retinal and vitreal retinopathy Skin Systemic sclerosis and scleroderma, keloids, hypertrophic scars, burns, genetic factors NFS Pancreas Autoimmune/hereditary causes Intestine Crohn's disease/inflammatory bowl disease Brain Alzheimer's disease, AIDS Bone marrow Cancer, ageing Multi-organ Surgical complications, chemotherapeutic drug-induced fibrosis, radiation- fibrosis induced fibrosis, mechanical injuries

A ‘fibrotic disease’ is any disease giving rise to fibrosis, whether as a main or a secondary symptom.

Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Fibrosis is characterized by the accumulation and reorganization of the extracellular matrix (ECM). Despite having obvious etiological and clinical distinctions, most chronic fibrotic disorders have in common a persistent irritant that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, especially collagens and proteoglycans, which progressively remodel and destroy normal tissue architecture ^(3, 4). Despite its enormous impact on human health, there are currently no approved treatments that directly target the mechanisms of fibrosis ⁵.

The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell.

Extracellular Matrix (ECM)

Fibrogenesis is a dynamic process involving complex cellular and molecular mechanisms that usually originates from tissue injury ⁶. Fibrogenesis is the result of an imbalance in normal ECM regulation that alters the concentration of macromolecules leading to increased tissue size and density, with progressively impaired function. These macromolecules are mainly fibrous proteins with structural and adhesive functions, such as collagens and proteoglycans.

Collagen

Collagens are widely distributed in the human body, i.e. ˜30% of the protein mass in the human body is composed of collagens. Collagens are responsible for the structural integrity of the ECM of most connective tissues. The ECM content results from a fine balance between synthesis and degradation tightly controlled through regulation of gene expression and protein secretion, but also through endogenous protease inhibition and protein degradation by metalloproteinases and cysteine proteases ⁷⁻⁹. Table 2 lists the major collagen types with their major tissue distribution.

TABLE 2 Major collagen types and their tissue distribution. Collagen type Tissue distribution I Most connective tissues II Cartilage, vitreous humor III Extensible connective tissues, e.g. liver, skin, lung, vascular system IV Basement membranes V Tissues containing collagen I VI Most connective tissues VII Skin, bladder, oral mucosa, umbilical cord, amnion VIII Many tissues, especially endothelium XIII Endothelial cells, skin, eye, heart, skeletal muscle XIV Vessel, bone, skin, cartilage, eye, nerve, tendon, uterus XXI Vessel, heart, stomach, kidney, skeletal muscle, placenta

Type I collagen is the most abundant collagen and is found in most connective tissues. It is especially important for the structure of bone and skin where the major collagenous components are type I and III collagens ¹⁰.

Collagen type I and III are the major components of liver and lung in a 1:1 ratio in healthy tissue. In addition, collagen type IV and VI are found in the basement membranes in most tissues. The most common localization of type V collagen is within the characteristic collagen fibrils, in association with the collagen type I and III ¹⁰.

Some collagens have a restricted tissue distribution: for example, type II, which is found almost exclusively in cartilage ¹¹.

During fibrogenesis the net amount of collagens increases¹²⁻¹⁴. Table 3 shows by way of example the collagen increase during liver fibrosis.

TABLE 3 Changes of the composition of collagen from normal to cirrhotic human liver ¹⁵. Collagen Collagen Distribution Distribution Collagen normal cirrhotic Times normal liver cirrhotic type Chains liver (mg/g) liver (mg/g) increased (%) liver (%) I α₁(I) α₂(I) 2 16 8 37 42 III α₁(III) 2 8 4 37 21 IV α₁(IV) 0.5 7 14 9 18 α₂(IV) V α₁(V) 0.9 7 8 17 18 α₂(V) α₃(V) VI α₁(VI) 0.01 0.1 10 0.2 0.3 α₂(VI) Elastin

Elastin is a protein present in many connective tissues, primarily those that are elastic. It has a very high content of the amino acids glycine, valine, alanine, and proline, and has a molecular weight of 64 to 66 kDa. It is organised in an irregular or random coil conformation made up of 830 amino acids. Elastin is made by linking many soluble tropoelastin protein molecules, in a reaction catalyzed by lysyl oxidase, to make a massive insoluble, durable cross-linked array.

Elastin serves an important function in arteries as a medium for pressure wave propagation to help blood flow and is particularly abundant in large elastic blood vessels such as the aorta. Elastin is also very important in the lungs, elastic ligaments and the skin.

Despite much efforts devoted to the understanding of elastin synthesis and turnover, neo-epitopes originating from the proteolytic cleavage of this matrix molecules have until now not been associated with disease development in fibrosis.

Vimentin

Vimentin is a member of the intermediate filament family of proteins. Intermediate filaments are an important structural feature of eukaryotic cells. They, along with microtubules and actin microfilaments, make up the cytoskeleton. Although most intermediate filaments are stable structures, in fibroblasts, vimentin exists as a dynamic structure. This filament is used as a marker for mesodermally derived tissues, and as such has been used as an immunohistochemical marker for sarcomas.

Hertig and coworkers (Hertig et al., J Am Soc Nephrol. 2008 August; 19(8):1584-91) investigated if epithelial-to-mesenchymal transition in renal tubular epithelial cells of subjects with chronic allograft nephropathy could predict the progression of fibrosis in the allograft and measured vimentin expression in 83 biopsies from these. They did find an association between elevated vimentin expression and the intestinal fibrosis score at 1 year after surgery.

In another study of hepatic fibrosis, Meriden and colleagues (Meriden et al., Clin Gastro & Hepatol 2010; 8:289-296) found a significant association between vimentin expression (in biopsies obtained at F0 stage) and fibrosis progression, with elevated levels predicting rapid progression of the hepatic fibrosis. Accordingly, we wanted to investigate if circulating fragments of vimentin could serve as sensitive and specific biomarkers of fibrosis.

Proteoglycans

Proteoglycans are a diverse group of macromolecules, which covalently link a variable number of glycosaminoglycan (GAG) side chains to a core protein ¹⁶. These GAGs are polymers of disaccharide repeats (e.g. N-acetyl glucosamine or N-acetyl galactosamine), which are acidic (negatively charged) due to hydroxyl, carboxylated and sulfated side groups on the disaccharide units. This makes them highly hydrophilic, thus aiding the diffusion of water and positive ions (e.g. sodium from extracellular fluids) ¹⁷. Furthermore, GAGs have the ability to form non-covalent links with for example hyaluronic acid chains to form even larger molecular complexes ¹⁶. Table 4 lists the most studied proteoglycans associated with connective tissue.

TABLE 4 Proteoglycans of the extracellular matrix of connective tissue Group Proteoglycans Origin Function Large extracellular Aggrecan ¹⁸ Articular cartilage Extracellular matrix stability proteoglycans (aggregating chondrocytes, intervertebral (hyaluronan binding) and hyaluronan-binding) disc, nasal cartilage Versican ^(19, 20) Connective tissue: fibroblast, Cell-cell and cell-matrix keratinocytes, smooth muscle interactions cells, mesangial cells Binding of sugars in Ca— dependent manner Neurocan ²¹ Nervous tissue Binds to neural cell adhesion molecules Brevican ²² Nervous tissue Extracellular matrix stability Small Leucine-rich Decorin ²³ Connective tissue, cartilage, Binds to and connect collagen proteoglycans (collagen- bone molecules (matrix stabilization binding) and thickness) Organogenesis Binding of TGFβ Biglycans ²⁴ Capillary endothelium, skin Cell differentiation (keratinocytes), epithelium of Binds and connect collagen kidney fibrils Fibromodulin Connective tissue, bone, Regulate orientation of collagen fibers ¹⁷ cartilage Lumican ²³ Cornea, muscle, cartilage, Controls spacing and kidney, lung, intestine thickness of collagen fibers Cell-associated Serglycins ²⁵ Widely distributed to Hemopoietic cell proteoglycans endothelium - intercellular differentiation compartments Adhesion and activation of lymphoid cells Syndecans ²⁶ Widely distributed - often cell Binds collagens, fibronectin, membrane bound thrombospondin, tenascin and bFGF Betaglycan ²⁷ Widely distributed TGFβ receptor and signaling Possible reservoir of TGFβ Basement membrane Perlecan ²⁸ All basement membranes Selective barrier for proteoglycans macromolecules Cell-adhesion C-Reactive Protein

C-reactive protein (CRP) is an acute phase serum protein produced by the liver in response to different clinical conditions such as, inflammation, infection, or trauma²⁹. The production of CRP is induced by cytokines such as IL-6, released from the affected or damaged tissues. The physiological role of CRP is yet unknown and discussions on its pro- or anti-inflammatory actions are ongoing.

Proteases

The imbalance between synthesis and degradation of ECM during fibrogenesis, results from conversion of the low-density subendothelial matrix into matrix rich in interstitial collagens. The increase in collagen and proteoglycans may be due to one or both of (1) a decrease in protein production and (2) impaired protein degradation, and hence less matrix degradation. The decreased protein degradation has recently received increased attention. In the regulation of this process matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play important roles, as well as other proteases and their inhibitors, such as cystein proteases and the cystatins.

MMPs

MMPs are a large group of endopeptidases, capable of degrading most if not all components of the ECM. Presently, more than 25 MMPs have been found. MMPs are characterized by an active site containing a metal atom, typically zinc, and are secreted as zymogens. Different MMPs are expressed in different tissues. In Table 5 MMPs in the liver are shown.

TABLE 5 MMPs in the liver³⁰⁻³² Family Protease Source Substrate Collagenases MMP-1 HSC I, II, III, VII, VIII, X, gelatin MMP-8 Neutrophil I, II, III, V, VII, X, gelatin MMP-13 HSC, MFB, KC I, II, III, VII, X, gelatin Stromelysins MMP-3 HSC III, IV, V, IX, X, XI, gelatin, laminin, fibronectin, proteoglycans, glycoproteins, elastin, pro-MMP-1/13 MMP-10 HSC III, IV, V, gelatin, elastin, aggrecan MMP-11 HC PAI-1, week activity against matrix proteins Gelatinases MMP-2 HSC, MBF I, II, III, IV, V, VII, X, XI, gelagin, elastin, laminin MMP-9 KC, HSC, HC I, II, III, IV, V, VII, X, XI, gelagin, elastin, laminin MMP-7 HSC Entactin, gelatin, elastin, fibronectin, vitronectin, laminin, fibrinogen Metalloelastase MMP-12 Macrophages Elastin, gelatins, IV, laminin, fibronectin, entactin, vitronectin, proteoglycan, myelin basic protein, α1-antitripsin MT-MMPs MMP-14 HSC, MFB, KC I, II, III, gelatin, fibronectin, vitronectin, laminin, fibrinogen, MMP-15 HC, BDEC pro-MMP-2, pro-MMP-13 Pro-MMP-2, fibronectin, tenascin, laminin, aggrecan, perlecan

TIMPs block MMPs' proteolytic activity by binding in a substrate- and tissue-specific manner to MMP and membrane-type 1 metalloproteinase in a trimolecular complex (Table 6). During fibrosis TIMP levels increase dramatically, and MMP levels increase modestly or remain relatively static (except MMP-2) which in all gives a decrease in degradation of collagens.

TABLE 6 TIMPs in the liver³¹ Name Sources Metalloproteinase inhibited TIMP-1 HSC, MFB, KC, HC Pro-MMP-9, MMP-1, MMP-2, MMP-3, MMP-13 TIMP-2 KC, HSC MT-MMP-1, MT-MMP-2, proMMP-2, MMP-3, MMP-13, MMP-7 TIMP-3 HC MT-MMP-1, MT-MMP-2, TACE, MMP-13 Fibroblast Activation Protein

Fibroblast Activation Protein alpha subunit (FAPa or FAP, alpha) is an integral membrane gelatinase belonging to the serine protease family. FAPa is the alpha subunit and DPP4 (CD26) the beta subunit of a heterodimeric membrane-bound proteinase complex also known as 170 kDa Melanoma Membrane Gelatinase, Integral Membrane Serine Proteinase and Seprase. Some cells make only FAPa homodimers, some only DPP4 homodimers. The monomer is inactive. FAP, alpha is selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas³³. This protein is thought to be involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis. It has been shown that expression of FAP increase with the stage of fibrosis^(34, 35).

Fibrosis Biomarkers

A number of biochemical markers have been suggested for fibrotic diseases, although not specific product of the disease. In Table 7 is an example of biochemical markers of liver fibrosis used in clinical trial. In addition there are a lot of examples of biomarkers of other fibrotic diseases^(12, 36-42).

TABLE 7 summarizes some of the known markers of liver fibrosis. Chronic liver Biomarker Parameters disease Reference One parameter CRP NASH ⁴³ Hyaluronan HCV ⁴⁴⁻⁴⁷ IGF-I HCV ⁴⁸ Leptin HCV ⁴⁹ PIIIP HCV ⁵⁰ Several parameters MP3 PIIINP, MMP1 HCV ^(51, 52) Zheng et al index HA, PIIICP, PIIINP, Laminin, C-IV Chronic ⁵³ hepatitis Lebensztjen et al Laminin-2, C-IV, MMP2, MMP9-TIMP1 index HBV ⁵⁴ index Tenascin, hyaluronana, Colalegn VI, TIMP-1 HBV ⁵⁵ Tsochatzis et al Leptin, adiponectin, resistin HCV, HBC, NASH ⁵⁶ index Patel et al index Hyaluronan, TIMP-1, α₂-macroglobulin HCV ⁵⁷ TIMP-1, tenascin, collagen IV, PIIINP, MMP2, NASH ⁵⁸ laminin, Hyaluronan Forns-index (76, 77) Age, platelet count, γGT, cholesterol HCV ^(51, 59-62) HIV/HCV FibroTest (76, 78) Haptoglobin, α₂-macroglobulin, apolipoprotein HCV ^(45, 51, 60, 61, 63-75) A1, γGT, bilirubin HIV/HCV NAFLD NAFLD in diabetes patients Actitest FibroTest + ALT HCV ^(65, 76-78) APRI (Wai-index) AST, platelet count HIV/HCV ^(45, 51, 60, 61, 64, 66, 79-87) HCV NAFLD Hepascore Bilirubin, γGT, hyaluronan, α₂-macroglobulin, age, HCV ^(51, 61, 64, 66, 88) gender HIV/HCV FIB-4 Platelet count, AST, ALT, age HIV/HCV ^(61, 83) SHASTA Hyaluronan, albumin, AST HIV/HCV ⁶¹ Fibroindex FORN + APRI HCV ⁸⁹ Fibrometer test Platelet count, prothrombin index, AST, α₂- HIV/HCV ^(51, 61, 64, 66, 81) macroglobulin, hyaluronan, urea, age HCV NAFLD NFSA Age, hyperglycaemia, body mass index, platelets, NAFLD ⁸¹ albumin, AST/ALT Ultrasound + APRI HCV ⁸² Metwally et al index Platelet count, albumin, AST, history of blood HCV ⁹⁰ transfusion, HBV core antibody Mohamadnejad et al Age, HBV DNA levels, alkaline phosphatase, HCV ⁹¹ index albumin, platelet counts, AST FibroSpect II Hyaluronan, TIMP-1, α₂-macroglobulin HCV ^(85, 92, 93) Stepwise Combination of APRI and Fibrotest HCV ⁹⁴ combination algorithms Imbert-Bismut index α₂ macroglobulin, AST, ALT γGT, total bilirubin, HCV ⁹⁵ albumin, α1 globulin, α₂ globulin, β globulin, γ globulin, apolipoprotein A₁ Nunes et al Age, Platelets, INR, CD4, AST/ALT, Hyaluronan, HCV/HIV ⁹⁶ YKL-40, PIIINP HCV Fibroscan +++ Fibroscan, Fibrotest, APRI, HCV ⁹⁷

U.S. Pat. No. 5,387,504 describes the neo-epitope VDIPEN released by the action of stromelysin at the aggrecan site N₃₄₁-F₃₄₂ and an RIA assay employing a monoclonal antibody specific for this neo-epitope. More generally the use of monospecific antibodies specific for fragments of aggrecan, generated by specific stromelysin cleavage are described. Elevations of stromelysin occur in osteoarthritis, rheumatoid arthritis, atherosclerotic lesions, gout, inflammatory bowel disease (IBD), idiopathic pulmonary fibrosis (IPF), certain cancers, joint injuries, and numerous inflammatory diseases. Stromelysin is reported to be elevated in idiopathic pulmonary fibrosis, and it is alleged that the assay can be conducted on blood or other biological fluids to detect stromelysin cleavage products of aggrecan and that quantitation of such fragments can be used diagnostically in respect of IPF as well as other conditions. However, no evidence for this is provided and there have to our knowledge been no subsequent publications validating this prediction. Such RIA assays have been commercially available for many years and no reports of their successful use in diagnosing or monitoring any fibrotic disease have appeared.

U.S. Pat. No. 7,225,080 discloses a method for diagnosis of an inflammatory, a fibrotic or a cancerous disease in a patient by measuring the values of at least four biochemical markers selected from the group consisting of α2-macroglobulin, AST (aspartate aminotransferase), ALT (alanine aminotransferase), GGT (gammaglutamyl transpeptidase), γ-globulin, total bilirubin, albumin, α1-globulin, α2-globulin, haptoglobin, β-globulin, apoA1, IL-10, TGF-β1, apoA2, and apoB in the serum or plasma of said patient, and subsequently combining said values in order to determine the presence of liver fibrosis and/or liver necroinflammatory lesions in said patient. The patent does not teach the quantitative measurement of peptide fragment carrying neo-epitopes generated during fibrotic disease.

U.S. Pat. No. 6,060,255 describes a method for diagnosing the degree of liver fibrosis, comprising the steps of measuring the concentration of type IV collagen high molecular weight form in a sample using an antibody that specifically binds to type IV collagen, and relating the measurement to the degree of liver fibrosis. Again, no use is made of neo-epitopes produced by proteolytic enzymes acting in the body. The sample is actually digested with pepsin, which may obscure the natural pattern of collagen cleavage in the sample.

U.S. Pat. No. 4,628,027 (Gay) discloses the production of antibodies specific for connective tissue proteins and, more particularly, the production of monoclonal antibodies by fused cell hybrids against human collagens and enzymes involved in collagen degradation. The use of monoclonal antibodies against connective tissue proteins to establish the collagen profile of histological, cytological and biological fluid samples is described. However, the patent does not describe the measurement of connective tissue proteins based on the binding of antibodies to neo-epitopes on said connective tissue proteins.

Guañabens N et al, J Bone Miner Res, 1998 ⁹⁸ evaluated the bone turnover markers N-telopeptide of type I collagen (NTX), C-telopeptide of type I collagen (CTX) and N-terminal pro-peptide of collagen type I (PINP) in patients with primary biliary cirrhosis, a disease with increased hepatic fibrosis. The level of NTX, CTX and PINP were elevated in patients compared to controls and correlated with the histological stage of the disease. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope JYDGKGVG⇓(SEQ ID NO2249). The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR⇓(SEQ ID NO2250). These markers are located in telopeptides of collagen type I and not in the internal part (the triple helical part) of collagen type I. The monoclonal antibodies employed for the PINP assay were raised against an internal epitope in the PINP sequence which is not a neo-epitope.

Møller S et al, Gut., 1999 ⁹⁹ demonstrated that the C-terminal cross linked telopeptide of type I collagen (ICTP) was elevated in alcoholic cirrhosis patients compared to controls. The study described showed that a biochemical marker can reflect hepatic fibrosis. The ICTP polyclonal antibody has been raised against trypsin and collagenase cleaved collagen type I. However, the antibodies are not binding to a neo-epitope.

Rosen H N et al, Calcif Tissue Int, 2004 ¹⁰⁰ assessed the bone turnover markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in women receiving hormone replacement treatment (HRT). In the study it was observed that the bone turnover markers decreased with treatment. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope JYDGKGVG⇓(SEQ ID NO2249). The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR⇓(SEQ ID NO2250). In contrast to the present invention, these antibodies were used for evaluation of bone metabolism and not fibrosis.

Lein M et al, Eur Urol, 2007 ¹⁰¹ evaluated the use of the neo-epitope specific bone turnover markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in prostate cancer patients receiving zoledronic acid. In the study it was observed that the bone turnover markers decreased with treatment. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope JYDGKGVG⇓(SEQ ID NO2249). The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR⇓(SEQ ID NO2250). In contrast to the present invention, these antibodies were used for evaluation of the bone metabolism during invasion of bone metastases and not fibrosis.

PIIINP has been used in a number of studies to assess the severity of fibrotic disease ¹⁰², in patients with skin fibrosis following severe burn trauma ¹⁰³, for disease progression in noncirrhotic primary biliary cirrhosis ¹⁰⁴, in primary biliary cirrhosis and chronic viral hepatitis C ¹⁰⁵.

PIIINP and ICTP were measured in patients with fibrosis of the myocardium ¹⁰⁶.

Many reports combine a set of biochemical markers to improve the predictive value of the biochemical index. Eleven different serum markers were measured in 205 patients with fibrotic staging from F0 to F4, and the most informative markers were alpha2 macroglobulin, alpha2 globulin (or haptoglobin), gamma globulin, apolipoprotein A1, gamma glutamyltranspeptidase, and total bilirubin ¹⁰⁷. An index of these markers had a negative predictive value (100% certainty of absence of F2, F3, or F4) was obtained for scores ranging from zero to 0.10 (12% [41] of all patients), and high positive predictive value (>90% certainty of presence of F2, F3, or F4) for scores ranging from 0.60 to 1.00 (34% [115] of all patients).

However, in none of the above mentioned reports is it suggested that measurements of peptide fragments based on antibodies binding to neo-epitopes as now claimed might be useful for the assessment of patients with fibrotic disease.

BRIEF SUMMARY OF THE INVENTION

The present invention now provides a method of diagnosis of fibrosis comprising, conducting an immunoassay to measure neo-epitope containing protein fragments naturally present in a patient biofluid sample, and associating an elevation of said measure in said patient above a normal level with the presence of fibrosis, wherein said immunoassay is conducted by a method comprising:

contacting protein fragments naturally present in said sample with an immunological binding partner reactive with a neo-epitope formed by cleavage of a protein by a proteinase and measuring the extent of binding of peptide fragments to said immunological binding partner to measure therein protein fragments comprising said neo-epitope, and wherein said protein is collagen type I, collagen type III, collagen type IV, collagen type V or collagen type VI, biglycan, decorin, lumican, versican, perlecan, neurocan, brevican, fibromodulin, serglycin, syndecan, betaglycan, CRP, or vimentin subject to the proviso that when the neo-epitopes are formed by cleavage of type I collagen, the cleavage is not at a site at which collagen type I is cleaved by cathepsin K. WO2009/059972 published on 14^(th) May 2009 (after the priority date hereof) discloses assays for neo-epitopes of collagen III, but does not disclose that an elevated level of such a measure is to be associated with the presence or extent of fibrosis. Optionally, an assay according to this invention is based on one of the proteins named above other than collagen Type III or if based on collagen Type III utilises an immunological binding partner against one of the neoepitopes formed at the cleavage sites PGIPGRNGDP* SEQ ID NO1, *ESCPTGPQNY SEQ ID NO2, or PKGDTGPRGP* SEQ ID NO3 (where * marks the cleavage site). For these purposes, cardiovascular disease may not be regarded as fibrosis, or the fibrosis detected according to the invention may be other than fibrosis accompanying cardiovascular disease. Optionally, an elevated result in an immunoassay according to this invention is associated with skin fibrosis, lung fibrosis, or liver fibrosis.

The method may comprise the preliminary step of obtaining a patient biofluid sample.

The invention includes a method of immunoassay to measure neo-epitope containing protein fragments naturally present in body fluid sample, wherein said immunoassay is conducted by a method comprising:

contacting protein fragments naturally present in said sample with an immunological binding partner reactive with a neo-epitope formed by cleavage of a protein by a proteinase and measuring the extent of binding of peptide fragments to said immunological binding partner to measure therein protein fragments comprising said neo-epitope, and wherein said protein is neurocan, brevican, fibromodulin, serglycin, syndecan, betaglycan, collagen type I, collagen type IV, collagen type V, collagen type VI, CRP, or vimentin subject to the proviso that when the neo-epitopes are formed by cleavage of type I collagen, the cleavage is not at a site at which collagen type I is cleaved by cathepsin K.

Optionally, an assay according to this invention is based on one of the proteins named above other than collagen Type III or if based on collagen Type III utilises an immunological binding partner against one of the neoepitopes formed at the cleavage sites PGIPGRNGDP* SEQ ID NO1, *ESCPTGPQNY SEQ ID NO2, or PKGDTGPRGP* SEQ ID NO3 (where * marks the cleavage site).

Said immunological binding partner may have specific binding affinity for peptide fragments comprising a C-terminal neoepitope or an N-terminal neoepitope.

Specific reactivity with or immunological affinity for a neo-epitope will imply that the relevant immunological binding partner is not reactive with intact protein from which the neo-epitope derives. Preferably, said immunological binding partner is not reactive with a neo-epitope sequence, such as a sequence listed below, if the sequence is prolonged past the respective cleavage site.

The term ‘immunological binding partner’ as used herein includes polyclonal and monoclonal antibodies and also specific binding fragments of antibodies such as Fab or F(ab′)₂. Thus, said immunological binding partner may be a monoclonal antibody or a fragment of a monoclonal antibody having specific binding affinity.

Preferably, said peptide fragments are fragments of Type I, III, IV, V, or VI collagen, elastin, C-reactive protein, or one of the proteoglycans Biglycan, Decorin, Versican, and Perlecan. The connective tissue proteins are preferred. Preferably, the neo-epitope sequence to which the immunological binding partner binds is not found in any other protein or is not found in any of the other proteins to which the method of the invention relates.

Several candidate proteases may be responsible for the digestion of proteins in the fibrotic tissues. Most likely, this is the result of the large range of complicated processes resulting in different neo-epitope profiles dependent on the levels of disease.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The invention will be further described and illustrated with reference to the following examples and the accompanying drawings, in which:

FIG. 1 shows a graph showing CO3 ELISA results of different biological samples: Pooled human serum samples (Serum); Human amniotic fluid (AF); Human fibroblast culture media (Fibr. Cltr.);

FIG. 2A shows a graph showing CO3 serum levels in sham operated (s) and bile duct ligated rats at baseline (b) and at termination (t);

FIG. 2B shows the corresponding delta-values of CO3 in rat serum: Termination levels—Baseline levels;

FIG. 3 shows a graph showing CO3 levels in different human serum samples. Normal serum: from healthy individuals. COPD: Chronic Obstructed Pulmonary Disease (leading to lung fibrosis). Scleroderma (leading to skin and lung fibrosis). HCV: Hepatitis virus C (leading to liver fibrosis);

FIG. 4A and FIG. 4B show Liver weights and Liver scores determined in Example 5. FIG. 4C shows Sirius Red photomicrographs showing the hepatic structure in sham-operated rats, and in BDL rats 2 and 4 weeks post-surgery;

FIG. 5 shows levels of MMP-9 cleavage fragments of Type III collagen measured according to the invention in Example 5. FIG. 5A shows MMP-9 mediated CO3 degradation serum levels in bile duct ligated (BDL)- or sham-operated rats. FIG. 5B shows CO3-610C delta values (termination-baseline paired), 2 weeks post-surgery P<0.0001 and 4 weeks post-surgery P=0.0016. FIG. 5C shows CTX-II levels in BDL- or sham-operated rats;

FIG. 6 shows levels of Type III collagen gene expression in BDL or sham-operated rats determined in Example 5;

FIG. 7 shows changes of expression levels of the MMP-9 cleavage fragment of Type III collagen reactive with the antibody used in Example 5 as determined by Western blot. FIG. 7A shows Western blot 2 and 4 weeks post-surgery and FIG. 7B shows bands from western blot quantified by densitometry;

FIG. 8 shows the results of histology staining of liver sections obtained in Example 5. FIG. 8A shows in the top row histology sections from BDL- or sham-operated rats stained with Sirius Red. The bottom row shows masked histology sections for quantifying total collagen content (red color) in the liver. FIG. 8B shows total collagen quantified by Visiopharm software—2 weeks post-surgery P=0.0081; 4 weeks post-surgery P=0.0047;

FIG. 9 shows correlations between measurements of fragments of Type III collagen according to the invention with other liver biomarkers as determined in Example 5 9. FIG. 9A shows a correlation of Col3a1 to CO3-610C was found with R²=0.6993, P<0.0001. FIG. 9B shows that a correlation of CO3-610C to % collagen was found with R²=0.2278 and P=0.0050. FIG. 9C shows that a correlation of Col3a1 to % collagen was found with R²=0.5409, P<0.0001;

FIG. 10 shows results obtained on human serum samples in Example 6; and

FIG. 11 shows results obtained in testing the reactivity of a monoclonal antibody recognizing an N-terminal neo-epitope from CRP;

FIG. 12 shows collagen accumulation in rat liver measured in Example 8;

FIG. 13 shows immunoassay results obtained in Example 8;

FIG. 14 shows the correlation of the immunoassay results of FIG. 13 with liver collagen content;

FIG. 15 shows a comparison of the results of an immunoassay according to the invention with measurements of hyaluronic acid and of Sirius red staining in Example 8;

FIG. 16 shows in the first panel the correlation of results from the immunoassay according to the invention with Sirius red staining and in the second panel the correlation between hyaluronic acid levels and Sirius red staining;

FIG. 17 shows the lack of correlation between the results of the immunoassay of the invention and hyaluronic acid levels;

FIG. 18 shows skin sections and skin thickness measurements described in Example 9. FIG. 18A shows a skin section from a PBS treated mouse at 8 weeks of treatment. FIG. 18B shows a skin section from Bleomycin treated mouse at 8 weeks of treatment. FIGS. 18C and 18D are plots of skin thickness increase between PBS (n=7/time point) and Bleomycin (n=13/time point) treated mice for 2 weeks (P=0.0029), 4 weeks (P=0.0004), 6 weeks (P<0.0001) and 8 weeks (P<0.0001);

FIG. 19 shows results from an immunoassay according to the invention in Example 9;

FIG. 20A shows in panel A Western Blot images obtained in Example 9 and in panel Figure B shows corresponding immunoassay results according to the invention;

FIG. 21 shows a correlation between immunoassay results and skin thickness measurements; and

FIG. 22 shows a correlation between urine immunoassay results and Western blot measurements described in Example 9

DETAILED DESCRIPTION OF THE INVENTION

Collagen Assays

Collagen Type I

We have determined that the enzymes listed in the following table cleave type I collagen at least the following cleavage sites (marked “.”):

TABLE 8 Collagen type I cleavage sites. Protease Collagen type I MMP-2 V.PGPMGPSGPRGLPGPPGAPGPQG.F SEQ ID NO 4 MMP-2 S.VPGPMGPSGPRGLPGPPGAPGPQG.F SEQ ID NO 5 MMP-2 G.ISVPGPMGPSGPRGLPGPPGAPGPQG.F SEQ ID NO 6 MMP-9 G.ISVPGPMGPSGPRGLPGPPGAPGPQG.F SEQ ID NO 6 MMP-13 G.FQGPPGEPGEPGASGPMGPRGPPGPPG.K SEQ ID NO 7 MMP-13 V.PGPMGPSGPRGLPGPPGAPGPQG.F SEQ ID NO 8 MMP-2 F.SGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRG.L SEQ ID NO 9 MMP-9 F.SGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRG.L SEQ ID NO 9 MMP-13 F.SGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRG.L SEQ ID NO 9 MMP-9 G.LPGERGRPGAPGPAG.A SEQ ID NO 10 MMP-13 G.LPGERGRPGAPGPAG.A SEQ ID NO 10 MMP-2 G.LTGSPGSPGPDGKTGPPGPAG.Q SEQ ID NO 11 MMP-2 E.RGSPGPAGPKGSPGEAGRPGEAGLPGAKG.L SEQ ID NO 12 MMP-2 G.ERGSPGPAGPKGSPGEAGRPGEAGLPGAKG.L SEQ ID NO 13 MMP-9 G.LTGSPGSPGPDGKTGPPGPAG.Q SEQ ID NO 14 MMP-9 G.LTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPG.A SEQ ID NO 15 MMP-9 G.LTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARG.Q SEQ ID NO 16 MMP-13 G.LTGSPGSPGPDGKTGPPGPAG.Q SEQ ID NO 14 MMP-13 G.ERGSPGPAGPKGSPGEAGRPGEAGLPGAKG.L SEQ ID NO 13 MMP-9 G.QDGRPGPPGPPGARG.Q SEQ ID NO 17 MMP-9 G.LTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARG.Q SEQ ID NO 18 MMP-2 G.KDGEAGAQGPPGPAGPAGERGEQGPAGSPGF.Q SEQ ID NO 19 MMP-2 G.ERGEQGPAGSPGF.Q SEQ ID NO 20 MMP-3 E.RGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGF.Q SEQ ID NO 21 MMP-8 E.RGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGF.Q SEQ ID NO 21 — 113 PKGDTGPRGP.122 SEQ ID NO 22 P indicates hydroxyproline, M indicates oxidised methionine, and K indicates hydroxylysine

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type I collagen, excluding cleavage at a cathepsin K type I collagen site.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

TABLE 9 N-terminal sequences  of protease generated peptide fragments of Collagen type I. (The symbol “.” Indicates   the cleavage site) Collagen I, alpha1 .ISVPGP SEQ ID NO 23 .LPGPGG SEQ ID NO 26 .ARGLPG SEQ ID NO 29 .LPGERG SEQ ID NO 32 .GGPPGP SEQ ID NO 35 .ERGSPG SEQ ID NO 38 .RGVPGP SEQ ID NO 41 .RGEQGP SEQ ID NO 44 .ARGAPG SEQ ID NO 47 .PIGNVG SEQ ID NO 50 .GADGPA SEQ ID NO 53 .QRGVVG SEQ ID NO 56 .PMGPPG SEQ ID NO 59 .LQGPPG SEQ ID NO 62 .FDFSF SEQ ID NO 65 .AKGEAG SEQ ID NO 68 .VQGPPG SEQ ID NO 71 .RGSPGP SEQ ID NO 74 .ARGQAG SEQ ID NO 77 .KDGEAG SEQ ID NO 80 .QGLPGP SEQ ID NO 83 .AGLPGP SEQ ID NO 86 .LAGPPG SEQ ID NO 89 .PSGASG SEQ ID NO 92 .AGQRGV SEQ ID NO 95 .VVGLPG SEQ ID NO 98 .TGDAGP SEQ ID NO 175 .VPGPMG SEQ ID NO 24 .FQGPPG SEQ ID NO 27 .SGLDGA SEQ ID NO 30 .VRGEPG SEQ ID NO 33 .NSGEPG SEQ ID NO 36 .LTGSPG SEQ ID NO 39 .VGPAGK SEQ ID NO 42 .PGERGV SEQ ID NO 45 .PGDRGE SEQ ID NO 48 .AAGRVG SEQ ID NO 51 .GPQGIA SEQ ID NO 54 .GLPGQR SEQ ID NO 57 .MGPPGL SEQ ID NO 60 .SAGAPG SEQ ID NO 63 .DFSF SEQ ID NO 66 .GIAGAP SEQ ID NO 69 .LPGPPG SEQ ID NO 72 .FAGPPG SEQ ID NO 75 .NVGAPG SEQ ID NO 78 .GEVGPP SEQ ID NO 81 .IAGQRG SEQ ID NO 84 .RGVVGL SEQ ID NO 87 .EPGKQG SEQ ID NO 90 .GKQGPS SEQ ID NO 93 .ARGPAG SEQ ID NO 96 .VGPPGP SEQ ID NO 99 .PGPMGP SEQ ID NO 25 .KNGDDG SEQ ID NO 28 .LDGAKG SEQ ID NO 31 .PGAKGA SEQ ID NO 34 .DGVAGP SEQ ID NO 37 .QDGRPG SEQ ID NO 40 .ERGEQG SEQ ID NO 43 .ANGAPG SEQ ID NO 46 .AKGDAG SEQ ID NO 49 .PPGPAG SEQ ID NO 52 .GQRGVV SEQ ID NO 55 .PGLPGP SEQ ID NO 58 .DKGETG SEQ ID NO 61 .RTGDAG SEQ ID NO 64 .ATGAAG SEQ ID NO 67 .IAGAPG SEQ ID NO 70 .AGPKGS SEQ ID NO 73 .QAGVMG SEQ ID NO 76 .PAGERG SEQ ID NO 79 .ARGERG SEQ ID NO 82 .LTGPIG SEQ ID NO 85 .AGPPGA SEQ ID NO 88 .ATGFPG SEQ ID NO 91 .GPPGPA SEQ ID NO 94 .ASGPAG SEQ ID NO 97 .GPPGPP SEQ ID NO 100

Alternatively, suitable immunological binding partners may be specifically reactive with any of the following sequences at the C terminal of a peptide:

TABLE 10 C-terminal sequences  of protease generated peptide fragments of Collagen type I (The symbol “.” Indicates   the cleavage site). Collagen I, alpha1 QLSYGY. SEQ ID NO 101 KGHRGF. SEQ ID NO 104 APGPAG. SEQ ID NO 107 GANGAP. SEQ ID NO 110 EPGPVG. SEQ ID NO 113 KGPAGE. SEQ ID NO 116 PPGPPG. SEQ ID NO 119 AVGPAG. SEQ ID NO 122 APGPDG. SEQ ID NO 125 KDGVRG. SEQ ID NO 128 PGPAGF. SEQ ID NO 131 SAGPPG. SEQ ID NO 134 GEVGPP. SEQ ID NO 81 PGPQGI. SEQ ID NO 140 IAGQRG. SEQ ID NO 84 GPSGEP. SEQ ID NO 146 PVGPVG. SEQ ID NO 149 EQGPSG. SEQ ID NO 152 GPPGPP. SEQ ID NO 100 QMGPRG. SEQ ID NO 161 PGADGQ. SEQ ID NO 164 PPGPKG. SEQ ID NO 167 PKGPAG. SEQ ID NO 170 GPAGRP. SEQ ID NO 173 TGPRGP. SEQ ID NO 177 EKSTGG. SEQ ID NO 102 PSGPRG. SEQ ID NO 105 FPGAVG. SEQ ID NO 108 ANGAPG. SEQ ID NO 46 EPGPTG. SEQ ID NO 114 RGSPGP. SEQ ID NO 74 PPGARG. SEQ ID NO 120 PAGPAG. SEQ ID NO 123 RGERGF. SEQ ID NO 126 PAGPTG. SEQ ID NO 129 EPGDAG. SEQ ID NO 132 ATGFPG. SEQ ID NO 91 GEKGSP. SEQ ID NO 138 GPQGIA. SEQ ID NO 54 GQRGVV. SEQ ID NO 55 ERGPPG. SEQ ID NO 147 PQGPRG. SEQ ID NO 150 PRGPPG. SEQ ID NO 153 GPPSAG. SEQ ID NO 156 PPGPAG. SEQ ID NO 52 PGPPGA. SEQ ID NO 162 AGSPGF. SEQ ID NO 165 PGERGA. SEQ ID NO 168 GRNGDP. SEQ ID NO 171 PPGPIG. SEQ ID NO 174 PPGPQG. SEQ ID NO 103 APGPQG. SEQ ID NO 106 SEGPQG. SEQ ID NO 109 SGPQGP. SEQ ID NO 112 RGFPGA. SEQ ID NO 115 LPGAKG. SEQ ID NO 118 PGKAGE. SEQ ID NO 121 AGPAGE. SEQ ID NO 124 PAGPRG. SEQ ID NO 127 TGARGA. SEQ ID NO 130 PAGPPG. SEQ ID NO 133 NAGPPG. SEQ ID NO 136 GAPGTP. SEQ ID NO 139 PQGIAG. SEQ ID NO 142 RGPPGP. SEQ ID NO 148 HRGFSG. SEQ ID NO 151 PPGPRG. SEQ ID NO 154 PPSAGF. SEQ ID NO 157 TPGPQG. SEQ ID NO 160 QGIAGQ. SEQ ID NO 163 LPGPSG. SEQ ID NO 166 PMGPPG. SEQ ID NO 59 SPGEQG. SEQ ID NO 172 TGDAGP. SEQ ID NO 175 Collagen Type III

We have determined that the enzymes listed in the following table cleave type III collagen at least the following cleavage sites (marked *):

TABLE 11 Cleavage sites in collagen type III. Protease Neo-Epitope MMP-1 A*GIPGAPGLMGARGPPGPA*G SEQ ID NO 178 MMP-1 K*GDPGPPGIPGRNGDPGI*P SEQ ID NO 179 MMP-1 G*LAGPPGMPGPRGSPGPQG*V SEQ ID NO 180 MMP-1 G*ERGLPGPPGIKGPAGIPGF*P SEQ ID NO 181 MMP-1 G*IAGITGARGLAGPPGMPGPR*G SEQ ID NO 182 MMP-1 G*IKGHRGFPGNPGAPGSPGPAG*Q SEQ ID NO 183 MMP-1 A*RGLAGPPGMPGPRGSPGPQGV*K SEQ ID NO 184 MMP-1 I*TGARGLAGPPGMPGPRGSPGPQG*V SEQ ID NO 185 MMP-1 I*TGARGLAGPPGMPGPRGSPGPQGV*K SEQ ID NO 186 MMP-1 G*ITGARGLAGPPGMPGPRGSPGPQG*V SEQ ID NO 187 MMP-1 G*VKGESGKPGANGLSGERGPPGPQG*L SEQ ID NO 188 MMP-1 G*SRGAPGPQGPRGDKGETGERGAAG*I SEQ ID NO 189 MMP-1 P*KGDAGQPGEKGSPGAQGPPGAPGPLG*I SEQ ID NO 190 MMP-1 G*ITGARGLAGPPGMPGPRGSPGPQGV*K SEQ ID NO 191 MMP-1 G*LRGGAGPPGPEGGKGAAGPPGPPGAAGTPG*L SEQ ID NO 192 MMP-1 G*HAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPG*L SEQ ID NO 193 MMP-1 A*GKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAG*I SEQ ID NO 194 MMP-1 G*LQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPG*L SEQ ID NO 195 MMP-3 G*ERGLPGPPGIKGPAGIPGF*P SEQ ID NO 196 MMP-3 A*VGGLAGYPGPAGPPGPPGPPGT*S SEQ ID NO 197 MMP-3 K*DGTSGHPGPIGPPGPRGNRGER*G SEQ ID NO 198 MMP-3 A*VGGLAGYPGPAGPPGPPGPPGTSGHPG*S SEQ ID NO 199 MMP-3 G*IAGITGARGLAGPPGMPGPRGSPGPQG*V SEQ ID NO 200 MMP-3 A*PGAPGGKGDAGAPGERGPPGLAGAPGLRG*G SEQ ID NO 201 MMP-3 A*VGGLAGYPGPAGPPGPPGPPGTSGHPGSPG*S SEQ ID NO 202 MMP-2 A*IGSPGPAGPRGPVGPSGPPG*K SEQ ID NO 203 MMP-3 + -8 G*AIGSPGPAGPRGPVGPSGPPG*K SEQ ID NO 204 MMP-8 P*AGQQGAIGSPGPA*G SEQ ID NO 205 MMP-8 G*GPPGVAGPPGGSGPAGPP*G SEQ ID NO 206 MMP-8 L*AGPPGMPGPRGSPGPQG*V SEQ ID NO 207 MMP-8 G*LSGERGPPGPQGLPGLA*G SEQ ID NO 208 MMP-8 R*GLAGPPGMPGPRGSPGPQG*V SEQ ID NO 209 MMP-8 G*LAGPPGMPGPRGSPGPQGV*K SEQ ID NO 210 MMP-8 R*GLAGPPGMPGPRGSPGPQGV*K SEQ ID NO 211 MMP-8 G*PQGPPGKNGETGPQGPPGP*T SEQ ID NO 212 MMP-8 G*VKGERGSPGGPGAAGFPGAR*G SEQ ID NO 213 MMP-8 A*RGLAGPPGMPGPRGSPGPQG*V SEQ ID NO 214 MMP-8 N*GLSGERGPPGPQGLPGLAGTA*G SEQ ID NO 215 MMP-8 A*VGGLAGYPGPAGPPGPPGPPGT*S SEQ ID NO 216 MMP-8 G*SPGGKGEMGPAGIPGAPGLMGA*R SEQ ID NO 217 MMP-8 T*GARGLAGPPGMPGPRGSPGPQG*V SEQ ID NO 218 MMP-8 V*KGESGKPGANGLSGERGPPGPQG*L SEQ ID NO 219 MMP-8 G*VKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAG*N SEQ ID NO 220 MMP-8 G*SPGAQGPPGAPGPLGIAGITGARGLAGPPG*M SEQ ID NO 221 MMP-8 R*GAPGEKGEGGPPGVAGPPGGSGPAGPPGPQ*G SEQ ID NO 222 MMP-8 R*GAPGEKGEGGPPGVAGPPGGSGPAGPPGPQ*G SEQ ID NO 223 MMP-8 G*IAGITGARGLAGPPGMPGPRGSPGPQG*V SEQ ID NO 224 MMP-9 G*IKGPAGIPGFPG*M SEQ ID NO 225 MMP-9 G*QPGVMGFPGPKG*N SEQ ID NO 226 MMP-9 G*IKGPAGIPGFPGMK*G SEQ ID NO 227 MMP-9 G*IKGPAGIPGFPGMKG*H SEQ ID NO 228 MMP-9 I*PGAPGLMGARGPPGPAG*A SEQ ID NO 229 MMP-9 G*ERGLPGPPGIKGPAGIP*G SEQ ID NO 230 MMP-9 G*IPGAPGLMGARGPPGPAG*A SEQ ID NO 231 MMP-9 G*FRGPAGPNGIPGEKGPAG*E SEQ ID NO 232 MMP-9 P*GIPGQPGSPGSPGPPGIC*E SEQ ID NO 233 MMP-9 G*ERGLPGPPGIKGPAGIPGF*P SEQ ID NO 234 MMP-9 A*VGGLAGYPGPAGPPGPPGPPG*T SEQ ID NO 235 MMP-9 G*VKGERGSPGGPGAAGFPGARG*L SEQ ID NO 236 MMP-9 G*DAGAPGAPGGKGDAGAPGERGPPG*L SEQ ID NO 237 MMP-9 Q*GPPGPTGPGGDKGDTGPPGPQGL*Q SEQ ID NO 238 MMP-9 G*INGSPGGKGEMGPAGIPGAPGLM*G SEQ ID NO 239 MMP-9 Q*GPPGEPGQAGPSGPPGPPGAIGPS*G SEQ ID NO 240 MMP-9 P*GPPGINGSPGGKGEMGPAGIPGAP*G SEQ ID NO 241 MMP-9 R*GLPGPPGSNGNPGPPGPSGSPGKDGPPGPAG*N SEQ ID NO 242 MMP-9 G*KNGETGPQGPPGPTGPGGDKGDTGPPGPQG*L SEQ ID NO 243 MMP-9 G*LPGIAGPRGSPGERGETGPPGPAGFPGAPG*Q SEQ ID NO 244 MMP-9 G*INGSPGGKGEMGPAGIPGAPGLMGARGPPGPAG*A SEQ ID NO 245 MMP-9 P*GINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAG*A SEQ ID NO 246 MMP-9 P*PGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPG*L SEQ ID NO 247 MMP-9 G*LKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAG*A SEQ ID NO 248 MMP-9 G*NTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLG*I SEQ ID NO 249 MMP-9 G*LMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRG*E SEQ ID NO 250 MMP-9 G*LRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKG*D SEQ ID NO 251 MMP-8 and -9 G*QQGAIGSPGPAGPRGPVGPSGPPG*K SEQ ID NO 252 MMP-9 K*GDPGPPGIPGRNGDPGIPGQPG*S SEQ ID NO 253 MMP-9 G*LRGGAGPPGPEGGKGAAGPPGPPG*A SEQ ID NO 254 MMP-9 G*KNGETGPQGPPGPTGPGGDKGDTGPPGPQG*L SEQ ID NO 255 MMP-9 G*YQGPPGEPGQAGPSGPPGPPG*A SEQ ID NO 256 MMP-9 G*VAGPPGGSGPAGPPGPQG*V SEQ ID NO 257 MMP-8, -9 and G*DKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAG*Q SEQ ID NO 258 -13 ADAMTS-5 Q*GHAGAQGPPGPPGIN*G SEQ ID NO 259 CathepsinK A*GERGAPGPA*G SEQ ID NO 260 CathepsinK A*GIPGFPGMK*G SEQ ID NO 261 CathepsinK F*PGMKGHRGFD*G SEQ ID NO 262 CathepsinK G*FPGARGLPGPPG*S SEQ ID NO 263 CathepsinK A*GFPGARGLPGPPG*S SEQ ID NO 264 CathepsinK P*PGPPGPPGTSGHP*G SEQ ID NO 265 CathepsinK G*FPGMKGHRGFD*G SEQ ID NO 266 CathepsinK Q*PGDKGEGGAPGLPGI*A SEQ ID NO 267 CathepsinK R*GDKGETGERGAAGIK*G SEQ ID NO 268 CathepsinK D*GRNGEKGETGAPGLK*G SEQ ID NO 269 CathepsinK A*GQPGDKGEGGAPGLPGIA*G SEQ ID NO 270 CathepsinK G*GPPGENGKPGEPGPKGD*A SEQ ID NO 271 CathepsinK A*GIPGFPGMKGHRGFD*G SEQ ID NO 272 CathepsinK R*GGAGEPGKNGAKGEPGPR*G SEQ ID NO 273 CathepsinK K*GERGSPGGPGAAGFPGARGLPGPP*G SEQ ID NO 274 CathepsinK I*PGVPGAKGEDGKDGSPGEPGANGLP*G SEQ ID NO 275 CathepsinK G*AAGFPGARGLPGPPGSNGNPGPPGPS*G SEQ ID NO 276 CathepsinK R*PGPPGPSGPRGQPGVMGFPGPKGN*D SEQ ID NO 277 CathepsinK Q*GPPGPPGINGSPGGKGEMGPAGIPGAP*G SEQ ID NO 278 CathepsinK A*GKDGESGRPGRPGERGLPGPPGIK*G SEQ ID NO 279 CathepsinK A*GARGNDGARGSDGQPGPPGPPGTAGFPG*S SEQ ID NO 280 CathepsinK S*PGVSGPKGDAGQPGEKGSPGAQGPPGAPG*P SEQ ID NO 281 CathepsinK R*GSDGQPGPPGPPGTAGFPGSPGAKGEVGPA*G SEQ ID NO 282 CathepsinK Q*GPPGPPGINGSPGGKGEMGPAGIPGAPGLM*G SEQ ID NO 283 CathepsinK A*GPPGPPGPPGTSGHPGSPGSPGYQGPPGEPG*Q SEQ ID NO 284 CathepsinK F*PGAPGQNGEPGGKGERGAPGEKGEGGPPGVA*G SEQ ID NO 285 CathepsinK A*GFPGAPGQNGEPGGKGERGAPGEKGEGGPPG*V SEQ ID NO 286 CathepsinK A*GARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPA*G SEQ ID NO 287 CathepsinK R*GAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPS*G SEQ ID NO 288 CathepsinS G*IAGITGARGL*A SEQ ID NO 289 CathepsinS A*GPPGPPGAAGTPGLQG*M SEQ ID NO 290 CathepsinS N*GLSGERGPPGPQGLPG*L SEQ ID NO 291 CathepsinS M*GARGPPGPAGANGAPGLR*G SEQ ID NO 292 CathepsinS N*GLSGERGPPGPQGLPGLA*G SEQ ID NO 293 CathepsinS G*IAGITGARGLAGPPGMPGPRG*S SEQ ID NO 294 CathepsinS G*IAGITGARGLAGPPGMPGPRGSPGPQG*V SEQ ID NO 295 CathepsinS R*GGAGPPGPEGGKGAAGPPGPPGAAGTPGLQ*G SEQ ID NO 296 CathepsinS S*GPKGDAGQPGEKGSPGAQGPPGAPGPLG*I SEQ ID NO 297 CathepsinS G*IAGITGARGLAGPPGMPGPRGSPGPQGVK*G SEQ ID NO 298 CathepsinS S*GPKGDAGQPGEKGSPGAQGPPGAPGPLG*I SEQ ID NO 299 CathepsinS G*IAGITGARGLAGPPGMPGPRGSPGPQGVK*G SEQ ID NO 300 CathepsinS A*VGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQ*G SEQ ID NO 301 CathepsinS E*PGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPG*L SEQ ID NO 302 ADAMTS1 I*PGFPGMKGHR*G SEQ ID NO 303 ADAMTS1 R*GSPGGPGAAGFPGAR*G SEQ ID NO 304 ADAMTS1 K*GPAGIPGFPGMKGHR*G SEQ ID NO 305 ADAMTS1 R*GLAGPPGMPGPRGSPGPQ*G SEQ ID NO 306 ADAMTS1 A*GITGARGLAGPPGMPGPR*G SEQ ID NO 307 ADAMTS1 L*GIAGITGARGLAGPPGMPGPR*G SEQ ID NO 308 ADAMTS1 T*GARGLAGPPGMPGPRGSPGPQ*G SEQ ID NO 309 ADAMTS1 Q*GPPGPPGINGSPGGKGEMGPAG*I SEQ ID NO 310 ADAMTS1 L*PGPPGIKGPAGIPGFPGMKGHR*G SEQ ID NO 311 ADAMTS1 A*GITGARGLAGPPGMPGPRGSPGPQ*G SEQ ID NO 312 ADAMTS1 T*GARGLAGPPGMPGPRGSPGPQGVK*G SEQ ID NO 313 ADAMTS1 R*GLPGPPGIKGPAGIPGFPGMKGHR*G SEQ ID NO 314 ADAMTS1 G*RPGLPGAAGARGNDGARGSDGQPGPPG*P SEQ ID NO 315 ADAMTS1 N*GAPGPMGPRGAPGERGRPGLPGAAGAR*G SEQ ID NO 316 ADAMTS1 A*GSRGAPGPQGPRGDKGETGERGAAGIK*G SEQ ID NO 317 ADAMTS1 R*GLAGPPGMPGPRGSPGPQGVKGESGKPGAN*G SEQ ID NO 318 ADAMTS1 R*GLAGPPGMPGPRGSPGPQGVKGESGKPGANGL*S SEQ ID NO 319 ADAMTS1 P*GPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGS*P SEQ ID NO 320 ADAMTS1 T*GARGLAGPPGMPGPRGSPGPQGVKGESGKPGAN*G SEQ ID NO 321 ADAMTS1 R*GAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER*G SEQ ID NO 322 ADAMTS1 G*GPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGF*P SEQ ID NO 323 ADAMTS1 K*SGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIK*G SEQ ID NO 324 ADAMTS4 I*PGFPGMKGHR*G SEQ ID NO 325 ADAMTS4 R*GLAGPPGMPGPR*G SEQ ID NO 326 ADAMTS4 G*PQGLQGLPGTGGPP*G SEQ ID NO 327 ADAMTS4 K*GPAGIPGFPGMKGHR*G SEQ ID NO 328 ADAMTS4 R*GLAGPPGMPGPRGSPGPQG*V SEQ ID NO 329 ADAMTS4 G*GPPGENGKPGEPGPKGDAGAP*G SEQ ID NO 330 ADAMTS4 A*PGFRGPAGPNGIPGEKGPAGER*G SEQ ID NO 331 ADAMTS4 E*KGSPGAQGPPGAPGPLGIAGITGAR*G SEQ ID NO 332 ADAMTS4 L*PGPPGIKGPAGIPGFPGMKGHR*G SEQ ID NO 333 ADAMTS4 R*GAPGFRGPAGPNGIPGEKGPAGER*G SEQ ID NO 334 ADAMTS4 R*GLPGPPGIKGPAGIPGFPGMKGHR*G SEQ ID NO 335 ADAMTS4 R*GPVGPSGPPGKDGTSGHPGPIGPPGPR*G SEQ ID NO 336 ADAMTS4 A*PGPQGPRGDKGETGERGAAGIKGHR*G SEQ ID NO 337 ADAMTS4 R*GAPGPQGPRGDKGETGERGAAGIKGHR*G SEQ ID NO 338 ADAMTS4 R*GFPGNPGAPGSPGPAGQQGAIGSPGPAGPR*G SEQ ID NO 339 ADAMTS4 L*PGPPGIKGPAGIPGFPGMKGHRGFDGR*N SEQ ID NO 340 ADAMTS4 D*AGQPGEKGSPGAQGPPGAPGPLGIAGITGAR*G SEQ ID NO 341 ADAMTS4 R*GPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPR*G SEQ ID NO 342 ADAMTS4 K*GDAGQPGEKGSPGAQGPPGAPGPLGIAGITGAR*G SEQ ID NO 343 ADAMTS4 R*NGEKGETGAPGLKGENGLPGENGAPGPMGPR*G SEQ ID NO 344 ADAMTS4 A*PGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGA*A SEQ ID NO 345 ADAMTS4 R*GAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPR*G SEQ ID NO 346 ADAMTS4 R*GSPGERGETGPPGPAGFPGAPGQNGEPGGKGER*G SEQ ID NO 347 ADAMTS4 G*HAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMG*A SEQ ID NO 348 ADAMTS4 R*GLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGER*G SEQ ID NO 349 ADAMTS8 L*GIAGITGARGL*A SEQ ID NO 350 ADAMTS8 I*PGFPGMKGHR*G SEQ ID NO 351 ADAMTS8 R*GLAGPPGMPGPR*G SEQ ID NO 352 ADAMTS8 Q*GPPGAPGPLGIAGITGAR*G SEQ ID NO 353 ADAMTS8 A*GITGARGLAGPPGMPGPR*G SEQ ID NO 354 ADAMTS8 A*GIPGAPGLMGARGPPGPAGAN*G SEQ ID NO 355 ADAMTS8 R*GLAGPPGMPGPRGSPGPQGVKG*E SEQ ID NO 356 ADAMTS8 K*GSPGAQGPPGAPGPLGIAGITGAR*G SEQ ID NO 357 ADAMTS8 L*PGPPGIKGPAGIPGFPGMKGHR*G SEQ ID NO 358 ADAMTS8 K*DGTSGHPGPIGPPGPRGNRGER*G SEQ ID NO 359 ADAMTS8 A*GITGARGLAGPPGMPGPRGSPGPQ*G SEQ ID NO 360 ADAMTS8 R*GLAGPPGMPGPRGSPGPQGVKGESG*K SEQ ID NO 361 ADAMTS8 R*GLAGPPGMPGPRGSPGPQGVKGESGKPGAN*G SEQ ID NO 362 ADAMTS8 R*GLAGPPGMPGPRGSPGPQGVKGESGKPGANGL*S SEQ ID NO 363 ADAMTS8 P*GPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGS*P SEQ ID NO 364 ADAMTS8 R*GAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGER*G SEQ ID NO 365 ADAMTS8 K*SGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGA*A SEQ ID NO 366 ADAMTS8 R*GAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGAR*G SEQ ID NO 367 MMP9 _*AIGPSG____*_ SEQ ID NO 368 MMP9 117′ PGIPGRNGDP*. 124′ SEQ ID NO 369 MMP9 142′ *ESCPTGPQNY 151′ SEQ ID NO 370 MMP9 113′ PKGDTGPRGP*.′122 SEQ ID NO 371

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type III collagen.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

TABLE 12 N-terminal sequences  of protease generated peptide fragments of  Collagen type III. Collagen type III GIPGAP SEQ ID NO 372 IAGITG SEQ ID NO 375 TGARGL SEQ ID NO 378 KGDAGQ SEQ ID NO 381 GKSGDR SEQ ID NO 383 DGTSGH SEQ ID NO 135 GPPGVA SEQ ID NO 158 GLSGER SEQ ID NO 387 GARGLA SEQ ID NO 111 GAPGEK SEQ ID NO 141 IPGAPG SEQ ID NO 117 GPPGPT SEQ ID NO 391 GLPGPP SEQ ID NO 394 GINGSP SEQ ID NO 397 LMGARG SEQ ID NO 400 DKGEPG SEQ ID NO 403 PGMKGH SEQ ID NO 406 FPGMKG SEQ ID NO 409 GQPGDK SEQ ID NO 412 GERGSP SEQ ID NO 415 GPPGPP SEQ ID NO 100 GFPGAP SEQ ID NO 420 IAGITG SEQ ID NO 375 GPPGSN SEQ ID NO 423 PGPQGH SEQ ID NO 426 SGDRGE SEQ ID NO 429 PGPPGI SEQ ID NO 432 GAPGFR SEQ ID NO 435 GSPGER SEQ ID NO 439 AIGPSG SEQ ID NO 368 GAPGPQ SEQ ID NO 445 GPTGPI SEQ ID NO 448 SRGAPG SEQ ID NO 451 AGQPGE SEQ ID NO 454 AGQQGA SEQ ID NO 457 GARGLA SEQ ID NO 111 GSRGAP SEQ ID NO 462 SPGAQG SEQ ID NO 465 GIPGQP SEQ ID NO 468 GDPGPP SEQ ID NO 373 IKGHRG SEQ ID NO 376 ITGARG SEQ ID NO 379 LRGGAG SEQ ID NO 382 LQGLPG SEQ ID NO 384 VGGLAG SEQ ID NO 155 AGPPGM SEQ ID NO 145 GLAGPP SEQ ID NO 388 KGESGK SEQ ID NO 374 QPGVMG SEQ ID NO 144 FRGPAG SEQ ID NO 137 INGSPG SEQ ID NO 392 KNGETG SEQ ID NO 395 PGENGK SEQ ID NO 398 YQGPPG SEQ ID NO 401 GHAGAQ SEQ ID NO 404 FPGARG SEQ ID NO 407 PGDKGE SEQ ID NO 410 GPPGEN SEQ ID NO 413 PGVPGA SEQ ID NO 416 GKDGES SEQ ID NO 418 NTGAPG SEQ ID NO 437 GLSGER SEQ ID NO 387 GPKGDA SEQ ID NO 424 PGFPGM SEQ ID NO 427 GITGAR SEQ ID NO 430 ESCPTG SEQ ID NO 433 RPGLPG SEQ ID NO 436 PQGLQG SEQ ID NO 440 PGFRGP SEQ ID NO 443 GFPGNP SEQ ID NO 446 GDAGQP SEQ ID NO 449 VAGPPG SEQ ID NO 452 PGAPGG SEQ ID NO 455 PGPPGP SEQ ID NO 458 GRNGEK SEQ ID NO 460 GGAGEP SEQ ID NO 463 PGVSGP SEQ ID NO 466 DAGAPG SEQ ID NO 469 LAGPPG SEQ ID NO 89 RGLAGP SEQ ID NO 377 VKGESG SEQ ID NO 380 ERGLPG SEQ ID NO 385 AIGSPG SEQ ID NO 143 LSGERG SEQ ID NO 176 IGSPGP SEQ ID NO 386 PQGPPG SEQ ID NO 389 VKGERG SEQ ID NO 159 IKGPAG SEQ ID NO 169 QQGAIG SEQ ID NO 390 GPPGEP SEQ ID NO 393 LPGIAG SEQ ID NO 396 LKGENG SEQ ID NO 399 GERGAP SEQ ID NO 402 GSDGQP SEQ ID NO 405 GFPGAR SEQ ID NO 408 GDKGET SEQ ID NO 411 GIPGFP SEQ ID NO 414 AAGFPG SEQ ID NO 417 GARGND SEQ ID NO 419 GAAGEP SEQ ID NO 421 GARGPP SEQ ID NO 422 GGAGPP SEQ ID NO 425 GSPGGP SEQ ID NO 428 GIAGIT SEQ ID NO 431 HAGAQG SEQ ID NO 434 GAPGPM SEQ ID NO 438 GPAGIP SEQ ID NO 441 KGSPGA SEQ ID NO 444 GPVGPS SEQ ID NO 447 NGEKGE SEQ ID NO 450 PGPQGP SEQ ID NO 453 PGAPGQ SEQ ID NO 456 SPGGKG SEQ ID NO 459 GPPGAP SEQ ID NO 461 GSPGAQ SEQ ID NO 464 PGAPGL SEQ ID NO 467 GPPGIN SEQ ID NO 470 or with any of the following sequences at the C-terminal of a peptide:

TABLE 13 C-terminal sequences   of protease generated peptide fragments of Collagen type III. Collagen type III GPPGPA SEQ ID NO 94 GMPGPR SEQ ID NO 473 ERGAAG SEQ ID NO 476 ERGPPG SEQ ID NO 147 PSGPPG SEQ ID NO 483 GLPGLA SEQ ID NO 486 GLAGTA SEQ ID NO 488 LAGPPG SEQ ID NO 89 IPGFPG SEQ ID NO 492 FPGAPG SEQ ID NO 494 GPPGIC SEQ ID NO 2187 PGPQGL SEQ ID NO 497 SPGPKG SEQ ID NO 499 TGAPGS SEQ ID NO 502 PGPKGD SEQ ID NO 506 GLPGIA SEQ ID NO 507 GLPGPP SEQ ID NO 394 IPGAPG SEQ ID NO 117 GEVGPA SEQ ID NO 514 GEVGPA SEQ ID NO 499 EKGPAG SEQ ID NO 515 TSGHPG SEQ ID NO 518 GTPGLQ SEQ ID NO 521 GPQGVK SEQ ID NO 524 PPGPAG SEQ ID NO 52 FPGMKG SEQ ID NO 409 GLSGER SEQ ID NO 387 GMKGHR SEQ ID NO 531 EMGPAG SEQ ID NO 534 GRNGDP SEQ ID NO 171 GVKGER SEQ ID NO 538 PQGVKG SEQ ID NO 541 GPPGPR SEQ ID NO 544 AGPRGA SEQ ID NO 547 GPAGAN SEQ ID NO 550 NGDPGI SEQ ID NO 471 SPGPAG SEQ ID NO 474 PGPLGI SEQ ID NO 477 PGPPGT SEQ ID NO 479 APGLRG SEQ ID NO 481 GSPGPA SEQ ID NO 484 GPPGPQ SEQ ID NO 490 FPGPKG SEQ ID NO 491 GPAGIP SEQ ID NO 441 PPGPPG SEQ ID NO 119 GAIGPS SEQ ID NO 495 LPGAAG SEQ ID NO 2188 GAPGLM SEQ ID NO 498 GEPGPR SEQ ID NO 500 GHRGFD SEQ ID NO 503 PGLPGI SEQ ID NO 504 PQGLPG SEQ ID NO 508 GANGLP SEQ ID NO 510 GPPGIK SEQ ID NO 512 PPGPQG SEQ ID NO 103 GFPGAR SEQ ID NO 408 EPGPRG SEQ ID NO 516 GAPGPA SEQ ID NO 519 GTSGHP SEQ ID NO 522 GAPGLK SEQ ID NO 525 PGPKGN SEQ ID NO 527 PGANGL SEQ ID NO 529 TGPRGP SEQ ID NO 177 EGGPPG SEQ ID NO 532 GIAGPR SEQ ID NO 535 GKPGAN SEQ ID NO 537 PGAAGF SEQ ID NO 539 GDAGAP SEQ ID NO 542 GPAGPR SEQ ID NO 545 GGKGER SEQ ID NO 548 SPGPQG SEQ ID NO 472 PGPQGV SEQ ID NO 475 AAGTPG SEQ ID NO 478 GNRGER SEQ ID NO 480 HPGSPG SEQ ID NO 482 GPAGPP SEQ ID NO 485 QGPPGP SEQ ID NO 487 PGLMGA SEQ ID NO 489 FPGARG SEQ ID NO 407 GFPGMK SEQ ID NO 493 AGIPGF SEQ ID NO 496 APGPLG SEQ ID NO 2189 GPPGIN SEQ ID NO 470 IPGQPG SEQ ID NO 501 LPGPPG SEQ ID NO 72 GAAGIK SEQ ID NO 505 GAPGLR SEQ ID NO 509 GPPGPS SEQ ID NO 511 TAGFPG SEQ ID NO 513 GPPGVA SEQ ID NO 158 TGARGL SEQ ID NO 378 PPGAPG SEQ ID NO 517 TPGLQG SEQ ID NO 520 MPGPRG SEQ ID NO 523 GSPGYQ SEQ ID NO 526 GAAGAR SEQ ID NO 528 GTGGPP SEQ ID NO 530 GITGAR SEQ ID NO 430 GSPGPQ SEQ ID NO 533 QPGPPG SEQ ID NO 536 VKGESG SEQ ID NO 380 TGERGA SEQ ID NO 540 GPAGER SEQ ID NO 543 RGFDGR SEQ ID NO 546 APGLMG SEQ ID NO 549 Collagen IV

We have determined that the enzymes listed in the following table cleave type IV collagen at least the following cleavage sites (marked “.”):

TABLE 14 Cleavage fragments of collagen type IV Protease Neo-Epitope FAP D.IDGYRGPPGP.Q SEQ ID NO 551 FAP S.MGPPGTPSVDHGF.L SEQ ID NO 552 FAP P.DGLPGSMGPPGTPSVDHG.F SEQ ID NO 553 FAP P.DGLPGSMGPPGTPSVDHGF.L SEQ ID NO 554 FAP P.DGLPGSMGPPGTPSVDHGFL.V SEQ ID NO 555 FAP P.SGRDGLPGPPGSPGPPGQPGY.T SEQ ID NO 556 FAP P.SGRDGLPGPPGSPGPPGQPGYTN.G SEQ ID NO 557 FAP P.SGRDGLPGPPGSPGPPGQPGYTNG.I SEQ ID NO 558 FAP I.PGSKGEQGFMGPPGPQGQPGLPGS.P SEQ ID NO 559 FAP P.RGFPGPPGPDGLPGSMGPPGTPSVD.H SEQ ID NO 560 FAP E.PGPPGLPGSVGSPG.V SEQ ID NO 561 FAP I.DGYRGPPGPQGP.P SEQ ID NO 562 FAP P.RGFPGPPGPDGLPGSMG.P SEQ ID NO 563 FAP D.GLPGSMGPPGTPSVDHGF.L SEQ ID NO 564 FAP D.GLPGSMGPPGTPSVDHGFL.V SEQ ID NO 565 FAP P.GLPGQQGAPGIPGFPGSKGEMGVMGTP.G SEQ ID NO 566 FAP I.GIPGMPGSPGLKGSPGSVGYPGSPGLPGE.K SEQ ID NO 567 FAP P.GPPGPPGEKGQMGLSFQGPKGDKGDQGVSGPPGVP.G SEQ ID NO 568 FAP P.GIGPPGARGPPGGQGPPGLSGPPGIKGEKGFPGFPGL.D SEQ ID NO 569 FAP E.PGLPGIPGVSGPK.G SEQ ID NO 570 FAP G.EKGQKGDTGPPGPPGLV.I SEQ ID NO 571 FAP L.PGIGVQGPPGPPGIPGPIGQPGLHGIPGEKGDPGPP.G SEQ ID NO 572 FAP G.SPGIPGHQGEMG.P SEQ ID NO 573 FAP E.PGMQGEPGPPGP.P SEQ ID NO 574 FAP G.PPGRLGAPGTPGLPGP.R SEQ ID NO 575 FAP P.PGPKGFPGIPGP.P SEQ ID NO 576 FAP A.KGQPGLPGFPGT.P SEQ ID NO 577 FAP D.RGPPGPPGIRGPPGP.P SEQ ID NO 578 FAP P.GPPGEKGKPGQDGIPGP.A SEQ ID NO 579 FAP L.LGSKGEKGEPGLPGIPGVSGPKGY.Q SEQ ID NO 580 MMP-9 D.GLPGSMGPPGTPSVDHG.F SEQ ID NO 581 MMP-9 D.GLPGSMGPPGTPSVDHGF.L SEQ ID NO 564 MMP-9 T.GPLGEKGERGYPGTPGPRGE.P SEQ ID NO 582 MMP-9 G.LQGIRGEPGPPGLPGSVGSPGVPGIGPPGARGPPGGQGPPG.L SEQ ID NO 583 MMP-9 P.DGLPGSMGPPGTPSVDHGFL.V SEQ ID NO 555 MMP-9 D.PGLKGDKGDVGLPGKPGSMDKVDMGS.M SEQ ID NO 584 MMP-9 L.PGPMGPPGLPGIDGV.K SEQ ID NO 585 MMP-9 D.GLPGSMGPPGTPSVDHGFL.V SEQ ID NO 565 MMP-9 G.IRGEPGPPGLPGSVGSPGVPGIGPPG.A SEQ ID NO 586 MMP-9 G.FPGPPGPDGLPGSMGPPGTPSVDHGF.L SEQ ID NO 587 MMP-9 G.LQGIRGEPGPPGLPGSVGSPGVPGIGPPG.A SEQ ID NO 588 MMP-9 G.IRGEPGPPGLPGSVGSPGVPGIGPPGARGPPGGQGPPG.L SEQ ID NO 589 MMP-9 E.DGVIGMMGFPGAIGP.P SEQ ID NO 590 MMP-9 Y.PGNPGILGPPGEDGVIGMMGFPGAIGPPGPPG.N SEQ ID NO 591 MMP-9 I.PPSDEICEPGPPGP.P SEQ ID NO 592 MMP-9 L.PGLPGPKGEPGLPGYPGNPGIKGS.V SEQ ID NO 593 MMP-9 G.IKGDKGSMGHPGPKGPP.G SEQ ID NO 594 MMP-9 T.PGSPGCAGSPGLPGSPGPPG.P SEQ ID NO 595 MMP-9 P.GAPGPQGLPGPPGFPGPVGPPGPPGFFGFPGAMGPRGPKGHMGE.R SEQ ID NO 596 MMP-9 G.LPGFAGNPGP SEQ ID NO 597 MMP-9 + FAP G.AEGLPGSPGFPGPQG.D SEQ ID NO 598 MMP-9 + FAP M.GPPGVPGFQGPKGLP.G SEQ ID NO 599 MMP-9 + FAP D.IDGYRGPPGPQGPPG.E SEQ ID NO 600 MMP-9 + FAP G.DQGDQGVPGAKGLPGP.P SEQ ID NO 601 MMP-9 + FAP G.DRGPQGQPGLPGLPGP.M SEQ ID NO 602 MMP-9 + FAP P.DGLPGSMGPPGTPSVDHGF.L SEQ ID NO 554 MMP-9 + FAP E.KGSIGIPGMPGSPGLKGSPGSVGYP.G SEQ ID NO 603 MMP-9 + FAP G.LQGIRGEPGPPGLPGSVGSPGVPGIGPPG.A SEQ ID NO 588 MMP-9 + FAP G.IRGEPGPPGLPGSVGSPGVPGIGPPGARGPPGGQGPPG.L SEQ ID NO 589 MMP-9 + FAP G.LQGIRGEPGPPGLPGSVGSPGVPGIGPPGARGPPGGQGPPG.L SEQ ID NO 583 MMP-9 + FAP G.LQGIRGEPGPPGLPGSVGSPGVPGIGPPGARGPPGGQGPPGLSGPPG.I SEQ ID NO 604 MMP-9 + FAP I.PPSDEICEPGPPGP.P SEQ ID NO 592 MMP-9 + FAP P.GPPGLMGPPGPPGLPGP.K SEQ ID NO 605 MMP-9 + FAP G.ERGSPGIPGAPGPIGPPGSPG.L SEQ ID NO 606 MMP-9 + FAP P.GIPGAPGAPGFPGSKGEPGDILTFPGMKGDKGELGSPGAPGL.P SEQ ID NO 607 MMP-9 + FAP C.DGGVPNTGPPGEPGPP.G SEQ ID NO 608 MMP12, Alpha1 .ILGHVPGML. SEQ ID NO 2190 MMP12, Alpha1 .PGLPGQPGPPGLPVPGQ. SEQ ID NO 2191 MMP12, Alpha1 .SGYPGNPGLPGIPGQDGPPGPPGIPGCNGTKGERGPLGPPGL. SEQ ID NO 2192 MMP12, Alpha1 .VSGPPGVPGQA. SEQ ID NO 2193 MMP12, Alpha1 .VSGPPGVPGQAQ. SEQ ID NO 2194 MMP12, Alpha2 .KRGPPGPPGLPGPPGPDGFL. SEQ ID NO 2195 MMP12, Alpha2 .LHGFPGAPGQEGPLG. SEQ ID NO 2196 MMP12, Alpha2 .LPGPDGPPGERGLPGEVL. SEQ ID NO 2197 MMP12, Alpha2 .LRGIPGF. SEQ ID NO 2198 MMP12, Alpha2 .PGFPGAPGTVGAPGIAGIPQK. SEQ ID NO 2199 MMP12, Alpha2 .QQGNRGLGF. SEQ ID NO 2200 MMP12, Alpha2 .VGQPGPNGIPSDTL. SEQ ID NO 2201 MMP12, Alpha3 .GEPGMQGEPGPPGPPGNLGPCGPRGKPGKDGKPGTPGPAGEKG. SEQ ID NO 2202 MMP12, Alpha3 .GEPGPPGPPGNLGPCGPRGKPGKDGKPGTPGPAGEKGNK. SEQ ID NO 2203 MMP12, Alpha3 .PGIPGTPGPPGLPGLQGPVGPPG. SEQ ID NO 2204 MMP12, Alpha3 .PGDIVFRK. SEQ ID NO 2205 MMP12, Alpha4 .GNKGDPASHFGPPGPKG. SEQ ID NO 2206 MMP12, Alpha4 .PGPRGKPGM. SEQ ID NO 2207 MMP12, Alpha5 .PGLPGQPGTRGL. SEQ ID NO 2208 MMP12, Alpha5 .PGPPGPLGIPGRSGVPGLKGDDGLQGQPGLPGPTGEKGSK. SEQ ID NO 2209 MMP12, Alpha5 .PGPPGPLGIPGRSGVPGLKGDDGLQGQPGLPGPTGEKGSKG. SEQ ID NO 2210 MMP12, Alpha5 .SKGEKGEPGLPGIPGVSGPKGYQGLPGDPGQPGLSGQPGL. SEQ ID NO 2211

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type IV collagen.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

TABLE 15 N-terminal sequences   of protease generated peptide fragments of Collagen type IV. Collagen type IV IDGYRG SEQ ID NO 609 PGSKGE SEQ ID NO 612 GPPGPP SEQ ID NO 100 LGSKGE SEQ ID NO 617 PGIGVQ SEQ ID NO 618 PGPKGF SEQ ID NO 621 PPSDEI SEQ ID NO 623 IRGEPG SEQ ID NO 626 DGVIGM SEQ ID NO 629 IKGDKG SEQ ID NO 631 GPPGVP SEQ ID NO 633 KGSIGI SEQ ID NO 635 ERGSPG SEQ ID NO 38 SGRDGL SEQ ID NO 639 DGYRGP SEQ ID NO 642 GIPGMP SEQ ID NO 645 .ILGHVP SEQ ID NO 2212 .PGLPGQ SEQ ID NO 2213 .SGYPGN SEQ ID NO 2217 .VSGPPG SEQ ID NO 2220 .KRGPPG SEQ ID NO 2223 .LHGFPG SEQ ID NO 2225 MGPPGT SEQ ID NO 610 RGFPGP SEQ ID NO 613 GLPGSM SEQ ID NO 615 GIGPPG SEQ ID NO 611 SPGIPG SEQ ID NO 619 KGQPGL SEQ ID NO 622 PGLKGD SEQ ID NO 624 FPGPPG SEQ ID NO 627 PGNPGI SEQ ID NO 630 PGSPGC SEQ ID NO 632 DQGDQG SEQ ID NO 634 PPGRLG SEQ ID NO 636 GIPGAP SEQ ID NO 372 GPPGEK SEQ ID NO 640 GPPGLM SEQ ID NO 643 PGPMGP SEQ ID NO 25 .LPGPDG SEQ ID NO 2214 .LRGIPG SEQ ID NO 760 .PGFPGA SEQ ID NO 2218 .QQGNRG SEQ ID NO 2221 .VGQPGP SEQ ID NO 2222 .GEPGMQ SEQ ID NO 2224 DGLPGS SEQ ID NO 611 PGPPGL SEQ ID NO 614 GLPGQQ SEQ ID NO 616 PGLPGI SEQ ID NO 504 PGMQGE SEQ ID NO 620 RGPPGP SEQ ID NO 148 GPLGEK SEQ ID NO 625 LQGIRG SEQ ID NO 628 PGLPGP SEQ ID NO 58 GAPGPQ SEQ ID NO 445 DRGPQG SEQ ID NO 442 EKGQKG SEQ ID NO 637 DGGVPN SEQ ID NO 638 AEGLPG SEQ ID NO 641 LPGFAG SEQ ID NO 644 .PGIPGT SEQ ID NO 2227 .PGDIVF SEQ ID NO 2215 .GNKGDP SEQ ID NO 2216 .PGPRGK SEQ ID NO 2219 .PGPPGP SEQ ID NO 458 .SKGEKG SEQ ID NO 2226 .GEPGPP SEQ ID NO 675 or with any of the following sequences at the C-terminal of a peptide:

TABLE 16 C-terminal sequences  of protease generated peptide fragments of  Collagen type IV. Collagen type IV RGPPGP SEQ ID NO 148 VDHGFL SEQ ID NO 648 PGLPGS SEQ ID NO 651 LPGSMG SEQ ID NO 654 PGLPGE SEQ ID NO 656 GPPGLV SEQ ID NO 658 PGLPGP SEQ ID NO 58 PGPRGE SEQ ID NO 663 GIGPPG SEQ ID NO 611 PGAIGP SEQ ID NO 668 GPKGPP SEQ ID NO 671 GSVGYP SEQ ID NO 673 GEPGPP SEQ ID NO 675 PGYTNG SEQ ID NO 678 LSGPPG SEQ ID NO 680 GVSGPK SEQ ID NO 682 HVPGML. SEQ ID NO 2228 GVPGQA. SEQ ID NO 2231 QEGPLG. SEQ ID NO 2234 NRGLGF. SEQ ID NO 2238 GEKGNK. SEQ ID NO 2241 PPGPKG. SEQ ID NO 167 GEKGSK. SEQ ID NO 2246 AGIPQK. SEQ ID NO 2237 SVDHGF SEQ ID NO 646 PGQPGY SEQ ID NO 649 GTPSVD SEQ ID NO 652 GPPGVP SEQ ID NO 633 GDPGPP SEQ ID NO 373 PGIPGP SEQ ID NO 659 DGIPGP SEQ ID NO 661 GQGPPG SEQ ID NO 664 PGIDGV SEQ ID NO 666 PPGPPG SEQ ID NO 119 SPGPPG SEQ ID NO 672 PQGPPG SEQ ID NO 389 AGNPGP SEQ ID NO 676 FPGPQG SEQ ID NO 679 PGAPGL SEQ ID NO 467 PGPPGP SEQ ID NO 458 LPVPGQ. SEQ ID NO 2229 VPGQAQ. SEQ ID NO 2232 LPGEVL. SEQ ID NO 2235 IPSDTL. SEQ ID NO 2239 PVGPPG. SEQ ID NO 2242 RGKPGM. SEQ ID NO 2244 EKGSKG. SEQ ID NO 2247 PSVDHG SEQ ID NO 647 QPGYTN SEQ ID NO 650 SVGSPG SEQ ID NO 653 PGFPGL SEQ ID NO 655 HQGEMG SEQ ID NO 657 PGFPGT SEQ ID NO 660 SGPKGY SEQ ID NO 662 KVDMGS SEQ ID NO 665 KGHMGE SEQ ID NO 667 KGLPGP SEQ ID NO 669 GPKGLP SEQ ID NO 670 PPGSPG SEQ ID NO 674 PGIKGS SEQ ID NO 677 PGPQGP SEQ ID NO 453 GVMGTP SEQ ID NO 681 LGPPGL. SEQ ID NO 2230 GPDGFL. SEQ ID NO 2233 RGIPGF. SEQ ID NO 2236 PAGEKG. SEQ ID NO 2240 DIVFRK. SEQ ID NO 2243 PGTRGL. SEQ ID NO 2245 SGQPGL. SEQ ID NO 2248 Collagen V

We have determined that the enzymes listed in the following table cleave type v collagen at least the following cleavage sites (marked “.” or in the absence of a ‘.’, at the end of the sequence):

TABLE 14A Cleavage fragments of collagen type V Protease Neo-epitope (COV) MMP2, Alpha3 K.GDPGPPGPIGSLG.H SEQ ID NO 683 MMP2, Alpha3 G.LRGIPGPVGEPG.L SEQ ID NO 684 MMP2, Alpha3 V.IGPPGLQGLPGPPGE.K SEQ ID NO 685 MMP2, Alpha3 G.KDGIPGPLGPLGPPG.A SEQ ID NO 686 MMP2, Alpha3 G.LRGIPGPVGEPGLL.G SEQ ID NO 687 MMP2, Alpha3 G.VLGPQGKTGEVGPLG.E SEQ ID NO 688 MMP2, Alpha3 K.DGIPGPLGPLGPPGAA.G SEQ ID NO 689 MMP2, Alpha3 G.EDGERGAEGPPGPTG.Q SEQ ID NO 690 MMP2, Alpha3 G.LQGPPGFPGPKGPPG.H SEQ ID NO 691 MMP2, Alpha3 P.IGSLGHPGPPGVAGPLG.Q SEQ ID NO 692 MMP2, Alpha3 G.IRGPPGTVIMMPFQ.F SEQ ID NO 693 MMP2, Alpha3 G.QMGPPGPLGPSGLPGLK.G SEQ ID NO 694 MMP2, Alpha3 G.LLGAPGQMGPPGPLGPSG.L SEQ ID NO 695 MMP2, Alpha3 G.LRGIPGPVGEPGLLGAPG.Q SEQ ID NO 696 MMP2, Alpha3 G.LLGPRGSPGPTGRPGVTG.I SEQ ID NO 697 MMP2, Alpha3 G.IRGPPGTVIMMPFQF.A SEQ ID NO 698 MMP2, Alpha3 G.KDGIPGPLGPLGPPGAAGP.S SEQ ID NO 699 MMP2, Alpha3 G.KDGIPGPLGPLGPPGAAGPSG.E SEQ ID NO 700 MMP2, Alpha3 Q.GLPGLEGREGAKGELGPPGPLG.K SEQ ID NO 701 MMP2, Alpha3 L.GPIGEKGKSGKTGQPGLEGERGPPGSRG.E SEQ ID NO 702 MMP2, Alpha3 G.LRGIPGPVGEPGLLGAPGQMGPPGPLGPSG.L SEQ ID NO 703 MMP2, Alpha3 G.ANGSPGERGPLGPAGGIGLPGQSGSEGPVGPAG.K SEQ ID NO 704 MMP2, Alpha3 G.LIGTPGEKGPPGNPGIPGLPGSDGPLGHPGHEGPTG.E SEQ ID NO 705 MMP2, Alpha1 G.LPGEPGPRG.L SEQ ID NO 706 MMP2, Alpha1 L.ALRGPAGPMG.L SEQ ID NO 707 MMP2, Alpha1 R.LALRGPAGPMG.L SEQ ID NO 708 MMP2, Alpha1 G.LTGRPGPVGPPGSGG.L SEQ ID NO 709 MMP2, Alpha1 G.LLGPKGPPGPPGPPG.V SEQ ID NO 710 MMP2, Alpha1 G.IPGRPGPQGPPGPAG.E SEQ ID NO 711 MMP2, Alpha1 P.GPDGPPGPMGPPGLP.G SEQ ID NO 712 MMP2, Alpha1 G.QPGPSGADGEPGPRG.Q SEQ ID NO 713 MMP2, Alpha1 G.ETGFQGKTGPPGPPG.V SEQ ID NO 714 MMP2, Alpha1 G.LRGFPGDRGLPGPV.G SEQ ID NO 715 MMP2, Alpha1 G.LRGFPGDRGLPGPVG.A SEQ ID NO 716 MMP2, Alpha1 G.KTGPIGPQGAPGKPGPDG.L SEQ ID NO 717 MMP2, Alpha1 G.PPGRPGLPGADGLPGPPG.T SEQ ID NO 718 MMP2, Alpha1 G.LKGNEGPPGPPGPAGSPGE.R SEQ ID NO 719 MMP2, Alpha1 G.LRGFPGDRGLPGPVGALG.L SEQ ID NO 720 MMP2, Alpha1 G.ERGHPGPPGPPGEQGLPG.L SEQ ID NO 721 MMP2, Alpha1 I.GPPGEQGEKGDRGLPGPQG.S SEQ ID NO 722 MMP2, Alpha1 G.EAGHPGPPGPPGPPGEVIQPLP.I SEQ ID NO 723 MMP2, Alpha1 K.PGPKGNSGGDGPAGPPGERGPNGP.Q SEQ ID NO 724 MMP2, Alpha1 G.EQGLPGSPGPDGPPGPMGPPGLPG.L SEQ ID NO 725 MMP2, Alpha1 E.GPPGEKGGQGPPGPQGPIGYPGPRG.V SEQ ID NO 726 MMP2, Alpha1 G.FPGPKGPPGPPGKDGLPGHPGQRG.E SEQ ID NO 727 MMP2 L.PFRFGGGGDA SEQ ID NO 728 MMP2 and 9 GSKGPMVSAQ.E SEQ ID NO 729 MMP2 and 9 Q.ESQAQAILQQ SEQ ID NO 730 MMP9, Alpha1 L.ALRGPAGPMG.L SEQ ID NO 707 MMP9, Alpha1 G.AIGPPGEKGPLG.K SEQ ID NO 731 MMP9, Alpha1 G.GPNGDPGPLGPPG.E SEQ ID NO 732 MMP9, Alpha1 P.PGPPGEQGLPGL.A SEQ ID NO 733 MMP9, Alpha1 G.LLGPKGPPGPPGPPG.V SEQ ID NO 734 MMP9, Alpha1 G.IPGRPGPQGPPGPAG.E SEQ ID NO 711 MMP9, Alpha1 G.QPGPSGADGEPGPRG.Q SEQ ID NO 713 MMP9, Alpha1 G.QQGNPGAQGLPGPQG.A SEQ ID NO 735 MMP9, Alpha1 G.KEGPPGEKGGQGPPG.P SEQ ID NO 736 MMP9, Alpha1 G.ETGFQGKTGPPGPPG.V SEQ ID NO 737 MMP9, Alpha1 G.EKGHPGLIGLIGPPG.E SEQ ID NO 738 MMP9, Alpha1 G.LRGFPGDRGLPGPVG.A SEQ ID NO 716 MMP9, Alpha1 G.KTGPIGPQGAPGKPGPDG.L SEQ ID NO 739 MMP9, Alpha1 P.GPDGPPGPMGPPGLPGLK.G SEQ ID NO 740 MMP9, Alpha1 G.ERGHPGPPGPPGEQGLPG.L SEQ ID NO 721 MMP9, Alpha1 G.ERGPNGPQGPTGFPGPKGPPGPPG.K SEQ ID NO 741 MMP9, Alpha1 L.IGLIGPPGEQGEKGDRGLPGPQGS.S SEQ ID NO 742 MMP9, Alpha1 E.GPPGEKGGQGPPGPQGPIGYPGPRG.V SEQ ID NO 726 MMP9, Alpha1 I.GPPGPPGLPGPPGPKGAKGSSGPTGPKGE.A SEQ ID NO 743 MMP9, Alpha1 P.LGPPGEKGKLGVPGLPGYPGRQGPKGSI.G SEQ ID NO 744 MMP9, Alpha1 Q.GPKGSIGFPGFPGANGEKGGRGTPGKPGPRG.Q SEQ ID NO 745 MMP9, Alpha3 P.GPKGDPGPPGPIG.S SEQ ID NO 746 MMP9, Alpha3 K.GDPGPPGPIGSLG.H SEQ ID NO 683 MMP9, Alpha3 A.PGIPGEKGLPGL.Q SEQ ID NO 747 MMP9, Alpha3 Q.GPPGPKGDPGPPGP.I SEQ ID NO 748 MMP9, Alpha3 G.SLGHPGPPGVAGPLG.Q SEQ ID NO 749 MMP9, Alpha3 G.KDGIPGPLGPLGPPG.A SEQ ID NO 686 MMP9, Alpha3 G.VLGPQGKTGEVGPLG.E SEQ ID NO 688 MMP9, Alpha3 G.ELGFQGQTGPPGPAG.V SEQ ID NO 750 MMP9, Alpha3 G.EDGERGAEGPPGPTG.Q SEQ ID NO 690 MMP9, Alpha3 G.LQGPPGFPGPKGPPG.H SEQ ID NO 691 MMP9, Alpha3 G.EKGHIGLIGLIGPPG.E SEQ ID NO 751 MMP9, Alpha3 G.QMGPPGPLGPSGLPGLK.G) SEQ ID NO 694 MMP9, Alpha3 G.PVGEPGLLGAPGQMGPPG.P SEQ ID NO 752 MMP9, Alpha3 G.LRGIPGPVGEPGLLGAPG.Q SEQ ID NO 696 MMP9, Alpha3 G.LLGPRGSPGPTGRPGVTG.I SEQ ID NO 697 MMP9, Alpha3 G.KDGIPGPLGPLGPPGAAGPSG.E SEQ ID NO 700 MMP9, Alpha3 Q.GLPGLEGREGAKGELGPPGPLG.K SEQ ID NO 701 MMP9, Alpha3 G.SRGERGPPGPTGKDGIPGPLGPLG.P SEQ ID NO 753 MMP9, Alpha3 G.EKGKSGKTGQPGLEGERGPPGSRG.E SEQ ID NO 754 MMP9, Alpha3 L.GPIGEKGKSGKTGQPGLEGERGPPGSRG.E SEQ ID NO 702 MMP9, Alpha3 G.ANGSPGERGPLGPAGGIGLPGQSGSEGPVGPAG.K SEQ ID NO 704 MMP9, Alpha3 G.LIGTPGEKGPPGNPGIPGLPGSDGPLGHPGHEGPTG.E SEQ ID NO 705 MMP13, Alpha1 L.PGEPGPRG.L SEQ ID NO 755 MMP13, Alpha1 A.LRGPAGPMG.L SEQ ID NO 756 MMP13, Alpha1 G.LPGEPGPRG.L SEQ ID NO 706 MMP13, Alpha1 L.ALRGPAGPMG.L SEQ ID NO 707 MMP13, Alpha1 R.LALRGPAGPMG.L SEQ ID NO 708 MMP13, Alpha1 G.LRGFPGDRGLPGPVG.A SEQ ID NO 716 MMP13, Alpha1 Q.ESQAQAILQQARLA.L SEQ ID NO 730 MMP13, Alpha1 P.GPDGPPGPMGPPGLPGLK.G SEQ ID NO 740 MMP13, Alpha1 G.PQGAIGPPGEKGPLGKPGLPGMPGADGPPGHPG.K SEQ ID NO 757 MMP13, Alpha1 A.GPMGLTGRPGPVGPPGSGGLKGEPGDVGPQGPRG.V SEQ ID NO 758 MMP13, Alpha3 G.VLGPQGKTGEVGPLG.E SEQ ID NO 688 MMP13, Alpha3 G.LRGIPGPVGEPGLLGAPG.Q SEQ ID NO 696 MMP13, Alpha3 G.LRGIPGPVGEPGLLGAPGQMGPPGPLGPSG.L SEQ ID NO 703 MMP13, Alpha3 G.LRGIPGPVGEPGLLGAPGQMGPPGPLGPSGLPG.L SEQ ID NO 759 P is hydroxyproline, K indicates hydroxylysine, glycosylation, lipoxidation or cross linking.

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type v collagen.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

TABLE 15a N-terminal sequences  of protease generated peptide fragments  of Collagen type V. Collagen type V GDPGPP SEQ ID NO 373 LQGPPG SEQ ID NO 62 ANGSPG SEQ ID NO 764 IPGRPG SEQ ID NO 767 LKGNEG SEQ ID NO 770 FPGPKG SEQ ID NO 491 LLGPKG SEQ ID NO 775 IGLIGP SEQ ID NO 778 GPPGPK SEQ ID NO 780 PVGEPG SEQ ID NO 783 ANGSPG SEQ ID NO 764 LLGAPG SEQ ID NO 785 LALRGP SEQ ID NO 788 LRGFPG SEQ ID NO 790 PGPKGN SEQ ID NO 527 AIGGPP SEQ ID NO 794 LLGPRG SEQ ID NO 797 GPKGDP SEQ ID NO 799 LQGPPG SEQ ID NO 62 SRGERG SEQ ID NO 803 LPGEPG SEQ ID NO 766 QMGPPG SEQ ID NO 802 ALRGPA SEQ ID NO 810 KDGIP SEQ ID NO 813 GPKGSI SEQ ID NO 816 LRGIPG SEQ ID NO 760 IGSLGH SEQ ID NO 762 LIGTPG SEQ ID NO 765 GPDGPP SEQ ID NO 768 ERGHPG SEQ ID NO 771 PFRFGG SEQ ID NO 773 QQGNPG SEQ ID NO 776 GPPGPP SEQ ID NO 100 SLGHPG SEQ ID NO 781 LRGIPG SEQ ID NO 760 LIGTPG SEQ ID NO 765 GLPGLE SEQ ID NO 786 LTGRPG SEQ ID NO 789 KTGPIG SEQ ID NO 791 EQGLPG SEQ ID NO 793 GPNGDP SEQ ID NO 795 GPDGPP SEQ ID NO 768 GDPGPP SEQ ID NO 373 EKGHIG SEQ ID NO 801 EKGKSG SEQ ID NO 804 PQGAIG SEQ ID NO 806 ETGFQG SEQ ID NO 808 EAGHPG SEQ ID NO 811 VLGPQG SEQ ID NO 814 ELGFQG SEQ ID NO 817 IGPPGI SEQ ID NO 761 IRGPPG SEQ ID NO 763 LPGEPG SEQ ID NO 766 QPGPSG SEQ ID NO 769 GPPGEQ SEQ ID NO 772 ESQAQA SEQ ID NO 774 KEGPPG SEQ ID NO 777 LGPPGE SEQ ID NO 779 KDGIPG SEQ ID NO 782 KDGIPG SEQ ID NO 782 PGEPGP SEQ ID NO 784 GIPGEK SEQ ID NO 787 LLGPKG SEQ ID NO 775 PPGRPG SEQ ID NO 792 GPPGEK SEQ ID NO 640 PGPPGE SEQ ID NO 796 ERGPNG SEQ ID NO 798 PGIPGE SEQ ID NO 800 QMGPPG SEQ ID NO 802 GPIGEK SEQ ID NO 805 GPMGLT SEQ ID NO 807 GSKGPM SEQ ID NO 809 EKGHPG SEQ ID NO 812 EDGERG SEQ ID NO 815 LRGPAG SEQ ID NO 818 P is hydroxyproline, K indicates hydroxylysine, glycosylation, lipoxidation or cross linking. or with any of the following sequences at the C-terminal of a peptide:

TABLE 16a C-terminal sequences  of protease generated peptide fragments   of Collagen type V. Collagen type V PIGSLG SEQ ID NO 819 PPGPTG SEQ ID NO 820 RPGVTG SEQ ID NO 823 PVGPAG SEQ ID NO 826 GPPGLP SEQ ID NO 828 AGSPGE SEQ ID NO 829 YPGPRG SEQ ID NO 831 LIGPPG SEQ ID NO 834 PPGPIG SEQ ID NO 174 QMGPPG SEQ ID NO 802 QQARLA SEQ ID NO 837 PLGPPG SEQ ID NO 839 IMMPFQ SEQ ID NO 842 AAGPSG SEQ ID NO 844 PAGPMG SEQ ID NO 847 GLPGPV SEQ ID NO 849 LPGPQG SEQ ID NO 852 PGPQGS SEQ ID NO 855 GPPGAA SEQ ID NO 857 PPGPAG SEQ ID NO 52 QGLPGL SEQ ID NO 859 PPGSRG SEQ ID NO 846 PPGPLG SEQ ID NO 845 PLGPLG SEQ ID NO 863 PVGEPG SEQ ID NO 783 PKGPPG SEQ ID NO 821 MMPFQF SEQ ID NO 824 HEGPTG SEQ ID NO 827 GQGPPG SEQ ID NO 664 PVGALG SEQ ID NO 830 HPGQRG SEQ ID NO 832 GLPGLK SEQ ID NO 835 KGLPGL SEQ ID NO 836 LLGAPG SEQ ID NO 785 PPGHPG SEQ ID NO 838 GEPGLL SEQ ID NO 840 GLPGLK SEQ ID NO 835 PPGPLG SEQ ID NO 845 PPGSGG SEQ ID NO 848 LPGPVG SEQ ID NO 850 VIQPLP SEQ ID NO 853 EKGPLG SEQ ID NO 856 TGPKGE SEQ ID NO 858 PPGPAG SEQ ID NO 52 PSGLPG SEQ ID NO 860 LPGPPG SEQ ID NO 72 KPGPRG SEQ ID NO 862 PPGSRG SEQ ID NO 846 PGPPGE SEQ ID NO 796 VAGPLG SEQ ID NO 822 PGAAGP SEQ ID NO 825 EPGPRG SEQ ID NO 516 PPGPPG SEQ ID NO 119 EQGLPG SEQ ID NO 793 GGGGDA SEQ ID NO 833 PPGPPG SEQ ID NO 119 PGPPGP SEQ ID NO 458 RPGVTG SEQ ID NO 823 PQGPRG SEQ ID NO 150 EVGPLG SEQ ID NO 841 PLGPSG SEQ ID NO 843 PPGSRG SEQ ID NO 846 PPGPPG SEQ ID NO 119 KPGPDG SEQ ID NO 851 RGPNGP SEQ ID NO 854 PLGPPG SEQ ID NO 839 GPKGSI SEQ ID NO 816 PPGPTG SEQ ID NO 820 LLGAPG SEQ ID NO 785 PPGLPG SEQ ID NO 861 PKGPPG SEQ ID NO 821 P is hydroxyproline, K indicates hydroxylysine, glycosylation, lipoxidation or cross linking. Collagen VI

We have determined that the enzymes listed in the following table cleave type vi collagen at least the following cleavage sites (marked “.” or in the absence of a ‘.’, at the end of the sequence):

TABLE 14B Cleavage fragments of collagen type VI Protease Neoepitope MMP2 G.YRGPEGPQGPPG.H SEQ ID NO 864 MMP2 G.PIGPKGYRGDEGPP.G SEQ ID NO 865 MMP2, I.GIGIGNADIT.E SEQ ID NO 866 (a3) MMP2, G.AQGPAGPAGPPG.L SEQ ID NO 867 (a3) MMP9 G.LIGEQGISGPRG.S SEQ ID NO 868 MMP9 P.PGLIGEQGISGPR.G SEQ ID NO 869 MMP9 E.PGEPGPKGGIGNRG.P SEQ ID NO 870 MMP9 G.ISGPRGSGGAAGAPGERGRTGPLG.R SEQ ID NO 871 MMP13 PGPAGPPGDPGLMG SEQ ID NO 872 FAP-1 VAAKPAAVRPPAAAAAKPVATKPEVPRP SEQ ID NO 873 FAP-1 GEPGLNGTTGPKGI SEQ ID NO 874 FAP-1 IGPKGIPGEDGYRGYPG SEQ ID NO 875 FAP-1 VAVVQHAPSESVDNASMPPVKVEFSL SEQ ID NO 876 FAP-2 LGPMGVPGRD SEQ ID NO 877 FAP-2 GEPGPPGEKGEAGDEGNPGPDGAPGERG SEQ ID NO 878 FAP-2 RGPIGSIGPKGIPGEDGYRGYPGDEGGP SEQ ID NO 879 FAP-2 PPPPQPARSAS SEQ ID NO 880 FAP-2 FGPSAATPAPPG SEQ ID NO 881 FAP-2 GPKGETGDLGPMGVPGRDGVPGGPGETGK SEQ ID NO 882

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neoepitope formed by cleavage of type v collagen.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

TABLE 15b N-terminal sequences  of protease generated peptide fragments   of Collagen type VI. Collagen type VI YRGPEG SEQ ID NO 883 ISGPRG SEQ ID NO 886 GEPGPP SEQ ID NO 675 AQGPAG SEQ ID NO 891 GEPGLN SEQ ID NO 894 FGPSAA SEQ ID NO 897 LGPMGV SEQ ID NO 899 PIGPKG SEQ ID NO 865 PGPAGP SEQ ID NO 887 RGPIGS SEQ ID NO 889 LIGEQG SEQ ID NO 892 IGPKGI SEQ ID NO 895 GPKGET SEQ ID NO 898 GIGIGN SEQ ID NO 885 VAAKPA SEQ ID NO 888 PPPPQP SEQ ID NO 890 PGLIGE SEQ ID NO 893 VAVVQH SEQ ID NO 896 PGEPGP SEQ ID NO 784 or with any of the following sequences at the C-terminal of a peptide:

TABLE 16b C-terminal sequences  of protease generated peptide fragments  of Collagen type VI. Collagen type VI GDEGPP SEQ ID NO 900 DPGLMG SEQ ID NO 902 GDEGGP SEQ ID NO 905 ISGPRG SEQ ID NO 886 YRGYPG SEQ ID NO 909 PGETGK SEQ ID NO 912 GNADIT SEQ ID NO 901 PEVPRP SEQ ID NO 903 PARSAS SEQ ID NO 906 GISGPR SEQ ID NO 907 KVEFSL SEQ ID NO 910 RTGPLG SEQ ID NO 913 PAGPPG SEQ ID NO 133 TGPKGI SEQ ID NO 904 TPAPPG SEQ ID NO 915 GIGNRG SEQ ID NO 908 GVPGRD SEQ ID NO 911 APGERG SEQ ID NO 914 Proteoglycans

In another aspect of the invention, said peptide fragments are fragments of proteoglycans versican, lumican, perlecan, biglycan and decorin, which are all identified in fibrotic tissue.

Several candidate proteases may be responsible for the digestion of proteoglycans in fibrotic lesions We have determined that the enzymes listed in table 17 generate lumican, versican, biglycan, perlecan and decorin resulting in at least following cleavage products:

TABLE 17 Cleavage fragments of biglycan, decorin, versican, lumican, and perlecan. Protease Biglycan MMP-3 SVPKEISPDTTLLDLQNNDISE SEQ ID NO 916 MMP-3 KSVPKEISPDTTLLDLQNNDISE SEQ ID NO 917 MMP-9 NSGFEPGAFDGLKLNYLRISEAK SEQ ID NO 918 MMP-9 LKSVPKEISPDTTLLDLQNNDISE SEQ ID NO 919 MMP-12 LRISEAKLTGIPKDLPET SEQ ID NO 920 MMP-13 LKSVPKEISPDTTLLDLQNNDISE SEQ ID NO 919 MMP-13 LTGIPKDLPETLNELHLDHNKIQAIE SEQ ID NO 921 ADAMTS4 RISEAKLTGIPKDLPETLNE SEQ ID NO 922 ADAMTS4 AIELEDLLRYSK SEQ ID NO 923 ADAMTS4 AIELEDLLRY SEQ ID NO 924 ADAMTS4 EAKLTGIPKDLPETLNE SEQ ID NO 925 ADAMTS4 LKAVPKEISPDTTLLDLQNNDISE SEQ ID NO 926 MMP-8 LLDLQNNDISELRKDD SEQ ID NO 927 MMP-8 IELEDLLRYS SEQ ID NO 928 CathepsinS NSGFEPGAFDGLK SEQ ID NO 929 Protease Decorin MMP-12 IVIELGTNPLK SEQ ID NO 930 MMP-3 DEASGIGPEVPDDR SEQ ID NO 931 MMP-3 LHLDGNKISRVDAAS SEQ ID NO 932 MMP-3 VNNKISKVSPGAFTPL SEQ ID NO 933 MMP-3 LILVNNKISKVSPGAFTPLVKLER SEQ ID NO 934 MMP-9 SNPVQYWEIQPSTFR SEQ ID NO 935 CathepsinK SSGIENGAFQGMK SEQ ID NO 884 CathepsinK SSGIENGAFQGMKKLS SEQ ID NO 946 ADAMTS1 KITEIKDGDFK SEQ ID NO 936 ADAMTS1 GLPPSLTELHLDGNK SEQ ID NO 937 Versican Unknown LLASDAGLYR SEQ ID NO 938 Unknown LATVGELQAAWR SEQ ID NO 939 Unknown ETTVLVAQNGNIK SEQ ID NO 940 Lumican Unknown SLEDLQLTHNK SEQ ID NO 941 Unknown LKEDAVSAAFK SEQ ID NO 942 Perlecan Unknown SIEYSPQLEDAGSR SEQ ID NO 943 Unknown LEGDTLIIPR SEQ ID NO 944 ADAMTS4 VSEAVVEKLEPEYR SEQ ID NO 945 ADAMTS4 EVSEAVVEKLEPEYR SEQ ID NO 947 ADAMTS4 SIEYSPQLEDASAKEFR SEQ ID NO 948

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of type versican, lumican, decorin, perlecan, and biglycan.

Suitable immunological binding partners may therefore be specifically reactive with any of the following at the N terminal of a peptide:

TABLE 18 N-terminal sequences of protease generated peptide fragments of biglycan, decorin, lumican, versican, and perlecan. Biglycan SVPKEI SEQ ID NO 949 NSGFEP SEQ ID NO 952 IELEDL SEQ ID NO 957 LRISEA SEQ ID NO 958 LLDLQN SEQ ID NO 961 GLKLNY SEQ ID NO 950 LKSVPK SEQ ID NO 953 QCSDLG SEQ ID NO 955 LTGIPK SEQ ID NO 959 RISEAK SEQ ID NO 951 AIELED SEQ ID NO 954 EAKLTG SEQ ID NO 956 LKAVPK SEQ ID NO 960 Decorin IVIELG SEQ ID NO 962 NGLNQM SEQ ID NO 965 SSGIEN SEQ ID NO 968 SNPVQY SEQ ID NO 971 DEASGI SEQ ID NO 963 LHLDGN SEQ ID NO 966 KITEIK SEQ ID NO 969 VNNKIS SEQ ID NO 964 LILVNN SEQ ID NO 967 GLPPSL SEQ ID NO 970 Versican LLASDA SEQ ID NO 972 ENQDAR SEQ ID NO 975 LATVGE SEQ ID NO 973 NGFDQC SEQ ID NO 976 ETTVLV SEQ ID NO 974 SLTVVK SEQ ID NO 977 Lumican SLEDLQ SEQ ID NO 978 LQHNRL SEQ ID NO 985 LKEDAV SEQ ID NO 979 HLQHNR SEQ ID NO 980 Perlecan SIEYSP SEQ ID NO 981 EVSEAV SEQ ID NO 984 LVNFTR SEQ ID NO 982 VSEAVV SEQ ID NO 983 or with any of the following sequences in table 19, at the C-terminal of a peptide:

TABLE 19 C-terminal sequences of  protease generated peptide fragments of biglycan, decorin, lumican, versican, and perlecan. Biglycan NNDISE SEQ ID NO 986 RISEAK SEQ ID NO 951 LRKDDF SEQ ID NO 991 KDLPET SEQ ID NO 994 LNELHL SEQ ID NO 997 YWEVQP SEQ ID NO 987 KIQAIE SEQ ID NO 989 LLRYSK SEQ ID NO 992 DLLRYS SEQ ID NO 995 EDLLRY SEQ ID NO 988 PETLNE SEQ ID NO 990 ELRKDD SEQ ID NO 993 AFDGLK SEQ ID NO 996 Decorin GTNPLK SEQ ID NO 998 SSGIEN SEQ ID NO 968 GMKKLS SEQ ID NO 1003 QPSTFR SEQ ID NO 1006 EVPDDR SEQ ID NO 999 RVDAAS SEQ ID NO 1001 KDGDFK SEQ ID NO 1004 AFQGMK SEQ ID NO 1007 GAFTPL SEQ ID NO 1000 LVKLER SEQ ID NO 1002 HLDGNK SEQ ID NO 1005 Versican CDVMYG SEQ ID NO 1008 IGQDYK SEQ ID NO 1010 NGFDQC SEQ ID NO 976 QNGINK SEQ ID NO 1009 Lumican QLTHNK SEQ ID NO 1011 VSAAFK SEQ ID NO 1012 GLKSLE SEQ ID NO 1013 Perlecan EDAGSR SEQ ID NO 1014 SAKEFR SEQ ID NO 1017 EFREVS SEQ ID NO 1015 LEPEYR SEQ ID NO 1018 VAQQDS SEQ ID NO 1016 CRP

Several candidate proteases may be responsible for the digestion of CRP in fibrotic tissue the literature reports many different proteases in fibrotic tissue. Most likely, this is the result of the large range of complicated processes eventually leading to fibrosis. However, in our assessment, early phases may consist of a range of MMPs, whereas later stages may rely more on cathepsin K degradation of the matrix, resulting in different neo-epitope profiles dependent on the levels of disease. We have through a range of in vitro cleavages of pure native proteins determined that the enzymes listed in the following tables of cleaved CRP at least following cleavage sites (marked * in Table 20, but at the ends of each sequence in Table 21):

TABLE 20 CRP fragments generated by specific proteases. Protease/Protein Neo-epitope CRP + CatK K*ESDTSYVSLKAPLT*K SEQ ID NO 1019 CRP + CatK G*GNFEGSQSLVGDIG*N SEQ ID NO 1020 CRP + MMP9 A*LKYEVQGEVFTKPQ*L SEQ ID NO 1021 CRP + MMP9 G*IVEFWVDGKPRV*R SEQ ID NO 1022 CRP + MMP1/MMP3 R*KAFVFPKE*S SEQ ID NO 1023 CRP + MMP3 K*YEVQGEVFTKPQLWP*- SEQ ID NO 1024 CRP + MMP3 D*SFGGNFEGSQS*L SEQ ID NO 1025 CRP + MMP3 D*FVLSPDEINT*I SEQ ID NO 1026 CRP + MMP3 S*LKKGYTVGAEA*S SEQ ID NO 1027 CRP + MMP3 A*FGQTDMSRKA*F SEQ ID NO 1028 CRP + MMP3 S*LKKGYTVGAEAS*I SEQ ID NO 1029 CRP + MMP3 G*EVFTKPQLWP*- SEQ ID NO 1030 CRP + MMP3 S*IILGQEQDSFGGNF SEQ ID NO 1031 CRP + MMP3 K*YEVQGEVFTKPQ.L SEQ ID NO 1032

TABLE 21 CRP fragments generated by specific proteases. Protease Neoepitope Aminoacid Nos* MMP9 AFVFPK SEQ ID NO 1033 026-031 MMP9 FGQTDMSR SEQ ID NO 1034 017-024 MMP9 FGQTDMSRK SEQ ID NO 1035 017-025 MMP9 FGQTDMSRKA SEQ ID NO 1036 017-026 MMP9 FGQTDMSRKAF SEQ ID NO 1037 017-027 MMP9 FGQTDMSRKAFVFPKE SEQ ID NO 1038 017-032 MMP9 FGQTDMSRKAFVFPKESDTS SEQ ID NO 1039 017-036 MMP9 FGQTDMSRKAFVFPKESDTSYV SEQ ID NO 1040 017-038 MMP9 FGQTDMSRKAFVFPKESDTSYVS SEQ ID NO 1041 017-039 MMP9 TDMSRKAFVFPKESDTSYV SEQ ID NO 1042 020-038 MMP9 MSRKAFVFPKESDTS SEQ ID NO 1043 022-036 MMP9 SRKAFVFPKESDTSYV SEQ ID NO 1044 023-038 MMP9 RKAFVFPKE SEQ ID NO 1045 024-032 MMP9 RKAFVFPKESDTSYV SEQ ID NO 1046 024-038 MMP9 RKAFVFPKESDTSYVS SEQ ID NO 1047 024-039 MMP9 KAFVFPKE SEQ ID NO 1048 025-032 MMP9 KAFVFPKESD SEQ ID NO 1049 025-034 MMP9 KAFVFPKESDT SEQ ID NO 1050 025-035 MMP9 KAFVFPKESDTS SEQ ID NO 1051 025-036 MMP9 KAFVFPKESDTSYV SEQ ID NO 1052 025-038 MMP9 KAFVFPKESDTSYVS SEQ ID NO 1053 025-039 MMP9 AFVFPKE SEQ ID NO 1054 026-032 MMP9 AFVFPKESDT SEQ ID NO 1055 026-035 MMP9 AFVFPKESDTSYV SEQ ID NO 1056 026-038 MMP9 AFVFPKESDTSYVS SEQ ID NO 1057 026-039 MMP9 AFVFPKESDTSYVSL SEQ ID NO 1058 026-040 MMP9 FVFPK SEQ ID NO 1059 027-031 MMP9 FVFPKE SEQ ID NO 1060 027-032 MMP9 FVFPKESD SEQ ID NO 1061 027-034 MMP9 FVFPKESDTS SEQ ID NO 1062 027-036 MMP9 FVFPKESDTSY SEQ ID NO 1063 027-037 MMP9 FVFPKESDTSYV SEQ ID NO 1064 027-038 MMP9 FVFPKESDTSYVS SEQ ID NO 1065 027-039 MMP9 FVFPKESDTSYVSL SEQ ID NO 1066 027-040 MMP9 VFPKESDTS SEQ ID NO 1067 028-036 MMP9 VFPKESDTSYV SEQ ID NO 1068 028-038 MMP9 VFPKESDTSYVS SEQ ID NO 1069 028-039 MMP9 VFPKESDTSYVSL SEQ ID NO 1070 028-040 MMP9 FPKESDTSYVS SEQ ID NO 1071 029-039 MMP9 KESDTSYVSLKAPLTKP SEQ ID NO 1072 031-047 MMP9 SDTSYVSLKAPLTKP SEQ ID NO 1073 033-047 MMP9 SLKAPLTKP SEQ ID NO 1074 039-047 MMP9 SLKAPLTKPLK SEQ ID NO 1075 039-049 MMP9 LKAPLTKPLK SEQ ID NO 1076 040-049 MMP9 FYTELSSTRGYS SEQ ID NO 1077 057-068 MMP9 LSSTRGYS SEQ ID NO 1078 061-068 MMP9 SSTRGYS SEQ ID NO 1079 062-068 MMP9 STRGYS SEQ ID NO 1080 063-068 MMP9 IFSYATKRQ SEQ ID NO 1081 069-077 MMP9 IFSYATKRQDNEILI SEQ ID NO 1082 069-083 MMP9 SYATKRQDNEILI SEQ ID NO 1083 071-083 MMP9 YATKRQDNEIL SEQ ID NO 1084 072-082 MMP9 YATKRQDNEILI SEQ ID NO 1085 072-083 MMP9 YATKRQDNEILIF SEQ ID NO 1086 072-084 MMP9 TKRQDNEILI SEQ ID NO 1087 074-083 MMP9 TKRQDNEILIF SEQ ID NO 1088 074-084 MMP9 TKRQDNEILIFWSKDI SEQ ID NO 1089 074-089 MMP9 KRQDNEILI SEQ ID NO 1090 075-083 MMP9 KRQDNEILIF SEQ ID NO 1091 075-084 MMP9 WSKDIGYS SEQ ID NO 1092 085-092 MMP9 SKDIGYS SEQ ID NO 1093 086-092 MMP9 IVEFWVDGKPRV SEQ ID NO 1094 124-135 MMP9 EFWVDGKPR SEQ ID NO 1095 126-134 MMP9 WVDGKPRV SEQ ID NO 1096 128-135 MMP9 VDGKPRV SEQ ID NO 1097 129-135 MMP9 SLKKGYTVGAE SEQ ID NO 1098 138-148 MMP9 SLKKGYTVGAEA SEQ ID NO 1099 138-149 MMP9 SLKKGYTVGAEAS SEQ ID NO 1100 138-150 MMP9 LKKGYTV SEQ ID NO 1101 139-145 MMP9 LKKGYTVG SEQ ID NO 1102 139-146 MMP9 LKKGYTVGA SEQ ID NO 1103 139-147 MMP9 LKKGYTVGAE SEQ ID NO 1104 139-148 MMP9 LKKGYTVGAEA SEQ ID NO 1105 139-149 MMP9 LKKGYTVGAEAS SEQ ID NO 1106 139-150 MMP9 LKKGYTVGAEASI SEQ ID NO 1107 139-151 MMP9 SIILGQEQDSFGGN SEQ ID NO 1108 150-163 MMP9 SIILGQEQDSFGGNFEGSQ SEQ ID NO 1109 150-168 MMP9 SIILGQEQDSFGGNFEGSQS SEQ ID NO 1110 150-169 MMP9 IILGQEQDSFGGNFEGS SEQ ID NO 1111 151-067 MMP9 IILGQEQDSFGGNFEGSQS SEQ ID NO 1112 151-169 MMP9 ILGQEQDSFGGN SEQ ID NO 1113 152-163 MMP9 ILGQEQDSFGGNFEGSQ SEQ ID NO 1114 152-168 MMP9 ILGQEQDSFGGNFEGSQS SEQ ID NO 1115 152-169 MMP9 LGQEQDSFGGNFEGSQ SEQ ID NO 1116 153-168 MMP9 LGQEQDSFGGNFEGSQS SEQ ID NO 1117 153-169 MMP9 GQEQDSFGGNFEGSQS SEQ ID NO 1118 154-169 MMP9 SFGGNFEGSQS SEQ ID NO 1119 159-169 MMP9 QSLVGDIGNVN SEQ ID NO 1120 168-178 MMP9 INTIYLGGPFSPNV SEQ ID NO 1121 189-202 MMP9 INTIYLGGPFSPNVLN SEQ ID NO 1122 189-204 MMP9 IYLGGPFSPNVLN SEQ ID NO 1123 192-204 MMP9 YLGGPFSPNVLN SEQ ID NO 1124 193-204 MMP9 LGGPFSPN SEQ ID NO 1125 194-201 MMP9 SPNVLNWRALKYEVQGEVFTKPQLWP SEQ ID NO 1126 199-224 MMP9 LNWRA SEQ ID NO 1127 203-207 MMP9 LNWRAL SEQ ID NO 1128 203-208 MMP9 LNWRALK SEQ ID NO 1129 203-209 MMP9 WRALKYE SEQ ID NO 1130 205-211 MMP9 WRALKYEV SEQ ID NO 1131 205-212 MMP9 WRALKYEVQGE SEQ ID NO 1132 205-215 MMP9 ALKYEV SEQ ID NO 1133 207-212 MMP9 LKYEVQ SEQ ID NO 1134 208-213 MMP9 LKYEVQG SEQ ID NO 1135 208-214 MMP9 LKYEVQGE SEQ ID NO 1136 208-215 MMP9 LKYEVQGEVFTKP SEQ ID NO 1137 208-220 MMP9 LKYEVQGEVFTKPQ SEQ ID NO 1138 208-221 MMP9 LKYEVQGEVFTKPQLWP SEQ ID NO 1139 208-224 MMP9 KYEVQGE SEQ ID NO 1140 209-215 MMP9 KYEVQGEVFTKPQ SEQ ID NO 1141 209-221 MMP9 KYEVQGEVFTKPQLWP SEQ ID NO 1142 209-224 MMP9 YEVQGEVFTKP SEQ ID NO 1143 210-220 MMP9 YEVQGEVFTKPQ SEQ ID NO 1144 210-221 MMP9 YEVQGEVFTKPQLWP SEQ ID NO 1145 210-224 MMP9 VQGEVFTKPQ SEQ ID NO 1146 212-221 MMP9 VQGEVFTKPQLWP SEQ ID NO 1147 212-224 MMP9 QGEVFTKPQ SEQ ID NO 1148 213-221 MMP9 GEVFTKP SEQ ID NO 1149 214-220 MMP9 GEVFTKPQ SEQ ID NO 1150 214-221 MMP9 EVFTKPQ SEQ ID NO 1151 215-221 MMP9 EVFTKPQLWP SEQ ID NO 1152 215-224 MMP9 VFTKPQ SEQ ID NO 1153 216-221 MMP9 FTKPQ SEQ ID NO 1154 217-221 MMP9 FTKPQLWP SEQ ID NO 1155 217-224 MMP9 TKPQLWP SEQ ID NO 1156 218-224 MMP9 KPQLWP SEQ ID NO 1157 219-224 MMP12 FGQTDMSRKA SEQ ID NO 1036 017-026 MMP12 MSRKAFVFP SEQ ID NO 1158 022-030 MMP12 MSRKAFVFPKE SEQ ID NO 1159 022-032 MMP12 MSRKAFVFPKESD SEQ ID NO 1160 022-034 MMP12 MSRKAFVFPKESDTS SEQ ID NO 1043 022-036 MMP12 MSRKAFVFPKESDTSYVS SEQ ID NO 1161 022-039 MMP12 SRKAFVFP SEQ ID NO 1162 023-030 MMP12 SRKAFVFPKESD SEQ ID NO 1163 023-034 MMP12 SRKAFVFPKESDTS SEQ ID NO 1164 023-036 MMP12 RKAFVFP SEQ ID NO 1165 024-030 MMP12 RKAFVFPKESD SEQ ID NO 1166 024-034 MMP12 KAFVFP SEQ ID NO 1167 025-030 MMP12 KAFVFPKE SEQ ID NO 1048 025-032 MMP12 KAFVFPKESD SEQ ID NO 1049 025-034 MMP12 AFVFPKE SEQ ID NO 1054 026-032 MMP12 AFVFPKESDTS SEQ ID NO 1168 026-036 MMP12 AFVFPKESDTSYVS SEQ ID NO 1057 026-039 MMP12 FVFPKE SEQ ID NO 1060 027-032 MMP12 FVFPKESD SEQ ID NO 1061 027-034 MMP12 FVFPKESDTS SEQ ID NO 1062 027-036 MMP12 FVFPKESDTSY SEQ ID NO 1063 027-037 MMP12 FVFPKESDTSYVS SEQ ID NO 1065 027-039 MMP12 VFPKESD SEQ ID NO 1169 028-034 MMP12 KESDTSY SEQ ID NO 1170 031-037 MMP12 KESDTSYVS SEQ ID NO 1171 031-039 MMP12 VSLKAP SEQ ID NO 1172 038-043 MMP12 LKAPLT SEQ ID NO 1173 040-045 MMP12 LKAPLTKP SEQ ID NO 1174 040-047 MMP12 YTELSSTRGYS SEQ ID NO 1175 058-068 MMP12 LSSTRGYS SEQ ID NO 1078 061-068 MMP12 STRGYS SEQ ID NO 1080 063-068 MMP12 YATKRQDNE SEQ ID NO 1176 072-080 MMP12 YATKRQDNEI SEQ ID NO 1177 072-081 MMP12 YATKRQDNEIL SEQ ID NO 1084 072-082 MMP12 TKRQDNEIL SEQ ID NO 1178 074-082 MMP12 KRQDNEIL SEQ ID NO 1179 075-082 MMP12 ILIFWSKD SEQ ID NO 1180 081-088 MMP12 IFWSKD SEQ ID NO 1181 083-088 MMP12 SKDIGYS SEQ ID NO 1093 086-092 MMP12 WVDGKPRV SEQ ID NO 1096 128-135 MMP12 WVDGKPRVR SEQ ID NO 1182 128-136 MMP12 VRKSLKKGYTVGAEAS SEQ ID NO 1183 135-150 MMP12 SLKKGYT SEQ ID NO 1184 138-144 MMP12 SLKKGYTVG SEQ ID NO 1185 138-146 MMP12 SLKKGYTVGA SEQ ID NO 1186 138-147 MMP12 SLKKGYTVGAE SEQ ID NO 1098 138-148 MMP12 SLKKGYTVGAEA SEQ ID NO 1099 138-149 MMP12 SLKKGYTVGAEAS SEQ ID NO 1100 138-150 MMP12 SLKKGYTVGAEASI SEQ ID NO 1187 138-151 MMP12 LKKGYTV SEQ ID NO 1101 139-145 MMP12 LKKGYTVG SEQ ID NO 1102 139-146 MMP12 LKKGYTVGA SEQ ID NO 1103 139-147 MMP12 LKKGYTVGAE SEQ ID NO 1104 139-148 MMP12 LKKGYTVGAEA SEQ ID NO 1105 139-149 MMP12 LKKGYTVGAEAS SEQ ID NO 1106 139-150 MMP12 LKKGYTVGAEASI SEQ ID NO 1107 139-151 MMP12 KKGYTVGAEAS SEQ ID NO 1188 140-150 MMP12 KGYTVGAEAS SEQ ID NO 1189 141-150 MMP12 KGYTVGAEASI SEQ ID NO 1190 141-151 MMP12 SIILGQEQDSFGGN SEQ ID NO 1108 150-163 MMP12 IILGQEQD SEQ ID NO 1191 151-158 MMP12 IILGQEQDSFGGN SEQ ID NO 1192 151-163 MMP12 IILGQEQDSFGGNFEGSQS SEQ ID NO 1112 151-169 MMP12 ILGQEQDSFGGN SEQ ID NO 1113 152-163 MMP12 LVGDIGNVNMWD SEQ ID NO 1193 170-181 MMP12 INTIYLGGPFSPNVLN SEQ ID NO 1122 189-204 MMP12 IYLGGPFSPN SEQ ID NO 1194 192-201 MMP12 IYLGGPFSPNV SEQ ID NO 1195 192-202 MMP12 IYLGGPFSPNVLN SEQ ID NO 1123 192-204 MMP12 LGGPFSPNVLN SEQ ID NO 1196 194-204 MMP12 WRALKYE SEQ ID NO 1130 205-210 MMP12 YEVQGEVFTKP SEQ ID NO 1143 210-220 MMP12 YEVQGEVFTKPQ SEQ ID NO 1144 210-221 MMP12 YEVQGEVFTKPQLWP SEQ ID NO 1145 210-224 MMP12 EVQGEVFTKP SEQ ID NO 1197 211-220 MMP12 EVQGEVFTKPQLWP SEQ ID NO 1198 211-224 MMP12 VQGEVFTKP SEQ ID NO 1199 212-220 MMP12 VQGEVFTKPQ SEQ ID NO 1146 212-221 MMP12 VQGEVFTKPQLWP SEQ ID NO 1147 212-224 MMP12 GEVFTKPQLWP SEQ ID NO 1200 214-224 MMP12 EVFTKP SEQ ID NO 1201 215-220 MMP12 EVFTKPQLWP SEQ ID NO 1152 215-224 MMP12 VFTKPQ SEQ ID NO 1153 216-221 MMP12 VFTKPQL SEQ ID NO 1202 216-222 MMP12 VFTKPQLWP SEQ ID NO 1203 216-224 MMP12 FTKPQLWP SEQ ID NO 1155 217-224 MMP12 TKPQLWP SEQ ID NO 1156 218-224 MMP1 AFVFPK SEQ ID NO 1033 006-031 MMP1 KAFVFPK SEQ ID NO 1204 025-031 MMP1 VRKSLK SEQ ID NO 1205 135-140 MMP1 YEVQGEVFTKPQLWP SEQ ID NO 1145 210-224 MMP3 FGQTDMSRKA SEQ ID NO 1036 017-026 MMP3 FGQTDMSRKAF SEQ ID NO 1037 017-027 MMP3 MSRKAFVFPKESDTSYV SEQ ID NO 1206 022-038 MMP3 MSRKAFVFPKESDTSYVS SEQ ID NO 1161 022-039 MMP3 SRKAFVFPKESDTSYV SEQ ID NO 1044 023-038 MMP3 SRKAFVFPKESDTSYVS SEQ ID NO 1207 023-039 MMP3 RKAFVFPKESDTSYV SEQ ID NO 1046 024-038 MMP3 RKAFVFPKESDTSYVS SEQ ID NO 1047 024-039 MMP3 KAFVFPKE SEQ ID NO 1048 025-032 MMP3 KAFVFPKESDTS SEQ ID NO 1051 025-036 MMP3 KAFVFPKESDTSYVS SEQ ID NO 1053 025-039 MMP3 KAFVFPKESDTSYVSL SEQ ID NO 1208 025-040 MMP3 KAFVFPKESDTSYVSLK SEQ ID NO 1209 025-041 MMP3 AFVFPKESDTSYVS SEQ ID NO 1057 026-039 MMP3 AFVFPKESDTSYVSL SEQ ID NO 1058 026-040 MMP3 AFVFPKESDTSYVSLKAP SEQ ID NO 1210 026-043 MMP3 FVFPKESDTSYV SEQ ID NO 1064 027-038 MMP3 FVFPKESDTSYVSLK SEQ ID NO 1211 027-041 MMP3 VFPKESDTSYVSLK SEQ ID NO 1212 028-041 MMP3 KESDTSYVSLKAP SEQ ID NO 1213 031-043 MMP3 TKRQDNEILIFW SEQ ID NO 1214 074-085 MMP3 IVEFWVDGKPRVRKS SEQ ID NO 1215 124-138 MMP3 SLKKGYTVGAEA SEQ ID NO 1099 138-149 MMP3 SLKKGYTVGAEAS SEQ ID NO 1100 138-150 MMP3 LKKGYTVGAEA SEQ ID NO 1105 139-149 MMP3 LKKGYTVGAEAS SEQ ID NO 1106 139-150 MMP3 LKKGYTVGAEASI SEQ ID NO 1107 139-151 MMP3 LKKGYTVGAEASII SEQ ID NO 1216 139-152 MMP3 SIILGQEQDSFGGNFEGSQS SEQ ID NO 1110 150-169 MMP3 IILGQEQDSFGGN SEQ ID NO 1192 151-163 MMP3 IILGQEQDSFGGNFEGSQS SEQ ID NO 1112 151-169 MMP3 ILGQEQDSFGGNFEGSQS SEQ ID NO 1115 152-169 MMP3 LGQEQDSFGGNFEGSQS SEQ ID NO 1117 153-169 MMP3 QEQDSFGGNFEGSQS SEQ ID NO 1217 155-169 MMP3 SFGGNFEGSQS SEQ ID NO 1119 159-169 MMP3 LVGDIGNVNMWD SEQ ID NO 1193 170-181 MMP3 FVLSPDEINT SEQ ID NO 1218 182-191 MMP3 YLGGPFSPNVLN SEQ ID NO 1124 193-204 MMP3 LKYEVQGEVFTKPQ SEQ ID NO 1138 208-221 MMP3 KYEVQGEVFTKPQ SEQ ID NO 1141 209-221 MMP3 KYEVQGEVFTKPQLWP SEQ ID NO 1142 209-224 MMP3 YEVQGEVFTKPQ SEQ ID NO 1144 210-221 MMP3 YEVQGEVFTKPQLWP SEQ ID NO 1145 210-224 MMP3 EVQGEVFTKPQLWP SEQ ID NO 1198 211-224 MMP3 VQGEVFTKPQLWP SEQ ID NO 1147 212-224 MMP3 GEVFTKPQLWP SEQ ID NO 1200 214-224 MMP3 EVFTKPQLWP SEQ ID NO 1152 215-224 MMP3 SKDIGYSFTVGGSEI SEQ ID NO 1219  86-100 MMP8 FGQTDMSR SEQ ID NO 1034 017-024 MMP8 FGQTDMSRK SEQ ID NO 1035 017-025 MMP8 FGQTDMSRKA SEQ ID NO 1036 017-026 MMP8 FGQTDMSRKAF SEQ ID NO 1037 017-027 MMP8 FGQTDMSRKAFV SEQ ID NO 1220 017-028 MMP8 FGQTDMSRKAFVFPKESDTSYV SEQ ID NO 1040 017-038 MMP8 MSRKAFVFPKESDTSYV SEQ ID NO 1206 022-038 MMP8 SRKAFVFPKESDTSYV SEQ ID NO 1044 023-038 MMP8 RKAFVFPKESDTSYV SEQ ID NO 1046 024-038 MMP8 KAFVFPKESDT SEQ ID NO 1050 025-035 MMP8 KAFVFPKESDTS SEQ ID NO 1051 025-036 MMP8 KAFVFPKESDTSYV SEQ ID NO 1052 025-038 MMP8 KAFVFPKESDTSYVS SEQ ID NO 1053 025-039 MMP8 AFVFPKESDTSYV SEQ ID NO 1056 026-038 MMP8 FVFPKESDTSYV SEQ ID NO 1064 027-038 MMP8 VFPKESDTSYV SEQ ID NO 1068 028-038 MMP8 FPKESDTSYV SEQ ID NO 1221 029-038 MMP8 SLKAPL SEQ ID NO 1222 039-044 MMP8 SLKAPLTKP SEQ ID NO 1074 039-047 MMP8 SLKAPLTKPLKA SEQ ID NO 1223 039-050 MMP8 RGYSIFSYA SEQ ID NO 1224 065-073 MMP8 FSYATKRQDNEILI SEQ ID NO 1225 070-083 MMP8 SYATKRQDNEILI SEQ ID NO 1083 071-083 MMP8 YATKRQDNEILI SEQ ID NO 1085 072-083 MMP8 ATKRQDNEILI SEQ ID NO 1226 073-083 MMP8 TKRQDNEILI SEQ ID NO 1087 074-083 MMP8 TKRQDNEILIF SEQ ID NO 1088 074-084 MMP8 FWSKDIGYS SEQ ID NO 1227 084-092 MMP8 FWSKDIGYSFT SEQ ID NO 1228 084-094 MMP8 FWSKDIGYSFTV SEQ ID NO 1229 084-095 MMP8 WSKDIGYSFTV SEQ ID NO 1230 085-095 MMP8 KSLKKGYTVGAEA SEQ ID NO 1231 137-149 MMP8 SLKKGYTVGAEA SEQ ID NO 1099 138-149 MMP8 LKKGYTV SEQ ID NO 1101 139-145 MMP8 LKKGYTVGAEA SEQ ID NO 1105 139-149 MMP8 LKKGYTVGAEAS SEQ ID NO 1106 139-150 MMP8 KKGYTVGAEA SEQ ID NO 1232 140-149 MMP8 GAEASIILGQE SEQ ID NO 1233 146-156 MMP8 GAEASIILGQEQD SEQ ID NO 1234 146-158 MMP8 SIILGQEQD SEQ ID NO 1235 150-158 MMP8 SIILGQEQDSFGGNFEGSQ SEQ ID NO 1109 150-168 MMP8 SIILGQEQDSFGGNFEGSQS SEQ ID NO 1110 150-169 MMP8 IILGQEQDSFGGN SEQ ID NO 1192 151-163 MMP8 IILGQEQDSFGGNFEGSQ SEQ ID NO 1236 151-168 MMP8 IILGQEQDSFGGNFEGSQS SEQ ID NO 1112 151-169 MMP8 ILGQEQDSFGGN SEQ ID NO 1113 152-163 MMP8 ILGQEQDSFGGNFEGS SEQ ID NO 1237 152-167 MMP8 ILGQEQDSFGGNFEGSQ SEQ ID NO 1114 152-168 MMP8 ILGQEQDSFGGNFEGSQS SEQ ID NO 1115 152-169 MMP8 LGQEQDSFGGN SEQ ID NO 1238 153-163 MMP8 LGQEQDSFGGNFEGS SEQ ID NO 1239 153-167 MMP8 LGQEQDSFGGNFEGSQ SEQ ID NO 1116 153-168 MMP8 LGQEQDSFGGNFEGSQS SEQ ID NO 1117 153-169 MMP8 LGQEQDSFGGNFEGSQSL SEQ ID NO 1240 153-170 MMP8 LGQEQDSFGGNFEGSQSLV SEQ ID NO 1241 153-171 MMP8 QDSFGGNFEGSQS SEQ ID NO 1242 157-169 MMP8 SFGGNFEGSQ SEQ ID NO 1243 159-168 MMP8 SFGGNFEGSQS SEQ ID NO 1119 159-169 MMP8 SFGGNFEGSQSLV SEQ ID NO 1244 159-171 MMP8 LVGDIGNVNMW SEQ ID NO 1245 170-180 MMP8 INTIYLGGPFSPN SEQ ID NO 1246 189-201 MMP8 TIYLGGPFSPN SEQ ID NO 1247 191-201 MMP8 IYLGGPFSPN SEQ ID NO 1194 192-201 MMP8 YLGGPFSPNV SEQ ID NO 1248 193-202 MMP8 YLGGPFSPNVLN SEQ ID NO 1124 193-204 MMP8 LGGPFSPNVLN SEQ ID NO 1196 194-204 MMP8 VLNWRA SEQ ID NO 1249 202-207 MMP8 VLNWRAL SEQ ID NO 1250 202-208 MMP8 VLNWRALK SEQ ID NO 1251 202-209 MMP8 LNWRAL SEQ ID NO 1128 203-208 MMP8 LNWRALK SEQ ID NO 1129 203-209 MMP8 LNWRALKYEV SEQ ID NO 1252 203-212 MMP8 NWRAL SEQ ID NO 1253 204-208 MMP8 NWRALKY SEQ ID NO 1254 204-210 MMP8 NWRALKYEV SEQ ID NO 1255 204-212 MMP8 NWRALKYEVQ SEQ ID NO 1256 204-213 MMP8 WRALKYE SEQ ID NO 1130 205-211 MMP8 WRALKYEVQ SEQ ID NO 1257 205-213 MMP8 WRALKYEVQGE SEQ ID NO 1132 205-215 MMP8 RALKYEV SEQ ID NO 1258 206-212 MMP8 RALKYEVQ SEQ ID NO 1259 206-213 MMP8 RALKYEVQGE SEQ ID NO 1260 206-215 MMP8 ALKYEV SEQ ID NO 1133 207-212 MMP8 ALKYEVQGEVFTKPQ SEQ ID NO 1261 207-221 MMP8 LKYEVQGE SEQ ID NO 1136 208-215 MMP8 LKYEVQGEVFTKPQ SEQ ID NO 1138 208-221 MMP8 KYEVQGEVFTKPQ SEQ ID NO 1141 209-221 MMP8 KYEVQGEVFTKPQLWP SEQ ID NO 1142 209-224 MMP8 YEVQGEVFTKPQ SEQ ID NO 1144 210-221 MMP8 YEVQGEVFTKPQLWP SEQ ID NO 1145 210-224 MMP8 EVQGEVFTKPQ SEQ ID NO 1262 211-221 MMP8 EVQGEVFTKPQLWP SEQ ID NO 1198 211-224 MMP8 VQGEVFTKPQ SEQ ID NO 1146 212-221 MMP8 VQGEVFTKPQLWP SEQ ID NO 1147 212-224 MMP8 QGEVFTKPQ SEQ ID NO 1148 213-221 MMP8 QGEVFTKPQL SEQ ID NO 1263 213-222 MMP8 QGEVFTKPQLWP SEQ ID NO 1264 213-224 MMP8 GEVFTKPQ SEQ ID NO 1150 214-221 MMP8 GEVFTKPQLWP SEQ ID NO 1200 214-224 MMP8 VFTKPQ SEQ ID NO 1153 216-221 MMP8 VFTKPQLWP SEQ ID NO 1203 216-224 MMP8 FTKPQLWP SEQ ID NO 1155 217-224 MMP8 TKPQLWP SEQ ID NO 1156 218-224 ADAMTS-1 ESDTSYVSLK SEQ ID NO 1265 032-041 ADAMTS-1 QEQDSFGGNFEGSQ SEQ ID NO 1266 155-168 ADAMTS-1 QEQDSFGGNFEGSQSLVG SEQ ID NO 1267 155-172 ADAMTS-1 GNFEGSQSLVG SEQ ID NO 1268 162-172 ADAMTS-1 YEVQGEVFT SEQ ID NO 1269 210-218 ADAMTS-1 YEVQGEVFTKPQ SEQ ID NO 1144 210-221 ADAMTS-1 GEVFTKPQ SEQ ID NO 1150 214-221 ADAMTS-8 VFPKESDTSYVS SEQ ID NO 1069 028-039 ADAMTS-8 QEQDSFGGNFEGSQSLVG SEQ ID NO 1267 155-172 ADAMTS-8 EINTIYL SEQ ID NO 1270 188-194 ADAMTS-8 KYEVQ SEQ ID NO 1271 209-213 ADAMTS-8 KYEVQGE SEQ ID NO 1140 209-215 Cat K FGQTDMSR SEQ ID NO 1034 017-024 Cat K AFVFPK SEQ ID NO 1033 026-031 Cat K FVFPK SEQ ID NO 1059 027-031 Cat K ESDTSYVSLK SEQ ID NO 1265 032-041 Cat K ESDTSYVSLKAPLT SEQ ID NO 1272 032-045 Cat K SDTSYVSLK SEQ ID NO 1273 033-041 Cat K DTSYVSLK SEQ ID NO 1274 034-041 Cat K STRGYS SEQ ID NO 1080 063-068 Cat K IFWSKDIG SEQ ID NO 1275 083-090 Cat K KGYTVGAE SEQ ID NO 1276 141-148 Cat K AEASIILGQEQDSFG SEQ ID NO 1277 147-161 Cat K LGQEQDSFG SEQ ID NO 1278 153-161 Cat K LGQEQDSFGGNFE SEQ ID NO 1279 153-165 Cat K GQEQDSFG SEQ ID NO 1280 154-161 Cat K GQEQDSFGGNFE SEQ ID NO 1281 154-165 Cat K GQEQDSFGGNFEGSQ SEQ ID NO 1282 154-168 Cat K GQEQDSFGGNFEGSQS SEQ ID NO 1118 154-169 Cat K QEQDSFGGN SEQ ID NO 1283 155-163 Cat K QEQDSFGGNFE SEQ ID NO 1284 155-165 Cat K QEQDSFGGNFEG SEQ ID NO 1285 155-166 Cat K QEQDSFGGNFEGS SEQ ID NO 1286 155-167 Cat K QEQDSFGGNFEGSQ SEQ ID NO 1266 155-168 Cat K QEQDSFGGNFEGSQS SEQ ID NO 1217 155-169 Cat K GNFEGSQSLV SEQ ID NO 1287 162-171 Cat K GNFEGSQSLVG SEQ ID NO 1268 162-172 Cat K GNFEGSQSLVGDIG SEQ ID NO 1288 162-175 Cat K GSQSLVGDIG SEQ ID NO 1289 166-175 Cat K GSQSLVGDIGNVN SEQ ID NO 1290 166-178 Cat K DFVLSPDEIN SEQ ID NO 1291 181-190 Cat K FVLSPDEINT SEQ ID NO 1218 182-191 Cat K VLSPDEINT SEQ ID NO 1291 183-191 Cat K GPFSPNVLN SEQ ID NO 1292 196-204 Cat K SPNVLNWR SEQ ID NO 1293 199-206 Cat K KYEVQG SEQ ID NO 1294 209-214 Cat K YEVQGEVFT SEQ ID NO 1269 210-218 Cat K YEVQGEVFTKPQ SEQ ID NO 1144 210-221 Cat K VQGEVFTKPQ SEQ ID NO 1146 212-221 Cat K GEVFTKPQ SEQ ID NO 1150 214-221 Cat K EVFTKPQ SEQ ID NO 1151 215-221 Cat S FGQTDMSR SEQ ID NO 1034 017-024 Cat S AFVFPKESDTSYVS SEQ ID NO 1057 026-039 Cat S FVFPKESDTSYVS SEQ ID NO 1065 027-039 Cat S VFPKESDTSYVS SEQ ID NO 1069 028-039 Cat S FPKESDTSYVS SEQ ID NO 1071 029-039 Cat S ESDTSYVSLK SEQ ID NO 1265 032-041 Cat S TSWESASGIVE SEQ ID NO 1295 116-126 Cat S KGYTVG SEQ ID NO 1296 141-146 Cat S QEQDSFGGNFE SEQ ID NO 1284 155-165 Cat S QEQDSFGGNFEG SEQ ID NO 1285 155-166 Cat S QEQDSFGGNFEGSQ SEQ ID NO 1266 155-168 Cat S QEQDSFGGNFEGSQS SEQ ID NO 1217 155-169 Cat S QEQDSFGGNFEGSQSLV SEQ ID NO 1297 155-171 Cat S QEQDSFGGNFEGSQSLVG SEQ ID NO 1267 155-172 Cat S SFGGNFEGSQSLVG SEQ ID NO 1298 159-172 Cat S GNFEGSQSLVG SEQ ID NO 1268 162-172 Cat S GNFEGSQSLVGDIG SEQ ID NO 1288 162-175 Cat S SPDEINTIYL SEQ ID NO 1299 185-194 Cat S SPDEINTIYLG SEQ ID NO 1300 185-195 Cat S LGGPFSPNVLN SEQ ID NO 1196 194-204 Cat S GGPFSPNVLN SEQ ID NO 1301 195-204 Cat S GPFSPNVLN SEQ ID NO 1292 196-204 Cat S ALKYE SEQ ID NO 1302 207-211 Cat S ALKYEVQ SEQ ID NO 1303 207-213 Cat S YEVQGEVF SEQ ID NO 1304 210-217 Cat S YEVQGEVFT SEQ ID NO 1269 210-218 Cat S YEVQGEVFTKPQ SEQ ID NO 1144 210-221 Cat S YEVQGEVFTKPQLWP SEQ ID NO 1145 210-224 Cat S VQGEVFTKPQLWP SEQ ID NO 1147 212-224 Cat S GEVFTKPQ SEQ ID NO 1150 214-221 Cat S GEVFTKPQLWP SEQ ID NO 1200 214-224 Cat S EVFTKPQLWP SEQ ID NO 1152 215-224 Cat S TKPQLWP SEQ ID NO 1156 218-224 Cat S KPQLWP SEQ ID NO 1157 219-224 *numbers in the sequence of CRP

Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of CRP by a protease at a site marked by the sign * in any one of the above partial sequences of CRP in Table 20 or at either end of any partial sequence of CRP in Table 21.

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of CRP.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

TABLE 22 N-terminal sequences of protease  generated peptide fragments of CRP. CRP AFVFPK SEQ ID NO 1033 VSLKAP SEQ ID NO 1172 TDMSRK SEQ ID NO 1307 VFPKES SEQ ID NO 1310 LSSTRG SEQ ID NO 1313 KRQDNE SEQ ID NO 1315 SIILGQ SEQ ID NO 1318 IYLGGP SEQ ID NO 1321 ALKYEV SEQ ID NO 1133 KPQLWP SEQ ID NO 1157 LVGDIG SEQ ID NO 1327 GAEASI SEQ ID NO 1330 EINTIY SEQ ID NO 1333 TSWESA SEQ ID NO 1336 YEVQGE SEQ ID NO 1339 RKAFVF SEQ ID NO 1342 TKPQLW SEQ ID NO 1345 SDTSYV SEQ ID NO 1347 IFSYAT SEQ ID NO 1348 EFWVDG SEQ ID NO 1351 LGQEQD SEQ ID NO 1354 SPNVLN SEQ ID NO 1357 QGEVFT SEQ ID NO 1358 IFWSKD SEQ ID NO 1181 FSYATK SEQ ID NO 1361 VLNWRA SEQ ID NO 1249 GSQSLV SEQ ID NO 1365 LKYEVQ SEQ ID NO 1134 GNFEGS SEQ ID NO 1369 FVFPKE SEQ ID NO 1060 FYTELS SEQ ID NO 1374 SFGGNF SEQ ID NO 1305 KAFVFP SEQ ID NO 1167 MSRKAF SEQ ID NO 1308 FPKESD SEQ ID NO 1311 SSTRGY SEQ ID NO 1314 WSKDIG SEQ ID NO 1316 IILGQE SEQ ID NO 1319 YLGGPF SEQ ID NO 1322 KYEVQG SEQ ID NO 1294 YTELSS SEQ ID NO 1325 QEQDSF SEQ ID NO 1328 QDSFGG SEQ ID NO 1331 DTSYVS SEQ ID NO 1334 SPDEIN SEQ ID NO 1337 FVLSPD SEQ ID NO 1340 IVEFWV SEQ ID NO 1343 EVQGEV SEQ ID NO 1346 SLKAPL SEQ ID NO 1222 SYATKR SEQ ID NO 1349 WVDGKP SEQ ID NO 1352 GQEQDS SEQ ID NO 1355 LNWRA SEQ ID NO 1127 GEVFTK SEQ ID NO 1359 VRKSLK SEQ ID NO 1205 ATKRQD SEQ ID NO 1362 NWRAL SEQ ID NO 1253 DFVLSP SEQ ID NO 1366 TKRQDN SEQ ID NO 1368 SLKKGY SEQ ID NO 1370 INTIYL SEQ ID NO 1372 WRALKY SEQ ID NO 1375 FGQTDM SEQ ID NO 1306 EVFTKP SEQ ID NO 1201 SRKAFV SEQ ID NO 1309 KESDTS SEQ ID NO 1312 STRGYS SEQ ID NO 1080 SKDIGY SEQ ID NO 1317 ILGQEQ SEQ ID NO 1320 LGGPFS SEQ ID NO 1323 VQGEVF SEQ ID NO 1324 ILIFWS SEQ ID NO 1326 RGYSIF SEQ ID NO 1329 TIYLGG SEQ ID NO 1332 AEASII SEQ ID NO 1335 GGPFSP SEQ ID NO 1338 LKKGYT SEQ ID NO 1341 ESDTSY SEQ ID NO 1344 FVFPK SEQ ID NO 1059 LKAPLT SEQ ID NO 1173 YATKRQ SEQ ID NO 1350 VDGKPR SEQ ID NO 1353 QSLVGD SEQ ID NO 1356 LNWRAL SEQ ID NO 1128 VFTKPQ SEQ ID NO 1153 KKGYTV SEQ ID NO 1360 FWSKDI SEQ ID NO 1363 NWRALK SEQ ID NO 1364 VLSPDE SEQ ID NO 1367 KGYTVG SEQ ID NO 1296 KSLKKG SEQ ID NO 1371 RALKYE SEQ ID NO 1373 GPFSPN SEQ ID NO 1376 or with any of the following sequences at the C-terminal of a peptide:

TABLE 23 C-terminal sequences  of protease generated peptide   fragments of CRP. CRP AFVFPK SEQ ID NO 1033 SPDEIN SEQ ID NO 1337 KESDTS SEQ ID NO 1312 LTKPLK SEQ ID NO 1379 KDIGYS SEQ ID NO 1380 GAEASI SEQ ID NO 1330 SPNVLN SEQ ID NO 1357 ALKYEV SEQ ID NO 1133 KAFVFP SEQ ID NO 1167 LKKGYT SEQ ID NO 1341 PRVRKS SEQ ID NO 1384 GYSFTV SEQ ID NO 1386 INTIYL SEQ ID NO 1372 NVLNWR SEQ ID NO 1389 VGAEAS SEQ ID NO 1392 QTDMSR SEQ ID NO 1394 PKESDT SEQ ID NO 1395 DNEILI SEQ ID NO 1397 YTVGAE SEQ ID NO 1400 FEGSQS SEQ ID NO 1403 LNWRA SEQ ID NO 1127 KYEVQG SEQ ID NO 1294 KRQDNE SEQ ID NO 1315 FTKPQL SEQ ID NO 1406 SLKAPL SEQ ID NO 1222 GSQSLV SEQ ID NO 1365 FGGNFE SEQ ID NO 1412 VQGEVF SEQ ID NO 1324 DMSRKA SEQ ID NO 1415 FPKESD SEQ ID NO 1311 KPQLWP SEQ ID NO 1157 DSFGGN SEQ ID NO 1378 SDTSYV SEQ ID NO 1347 STRGYS SEQ ID NO 1080 DGKPRV SEQ ID NO 1381 QGEVFT SEQ ID NO 1358 GPFSPN SEQ ID NO 1376 YEVQGE SEQ ID NO 1339 VSLKAP SEQ ID NO 1172 LGQEQD SEQ ID NO 1354 TVGSEI SEQ ID NO 1385 IILGQE SEQ ID NO 1319 WSKDIG SEQ ID NO 1316 ASGIVE SEQ ID NO 1390 APLTKP SEQ ID NO 1393 TDMSRK SEQ ID NO 1307 TSYVSL SEQ ID NO 1396 QDNEIL SEQ ID NO 1398 TVGAEA SEQ ID NO 1401 GNFEGS SEQ ID NO 1369 LNWRAL SEQ ID NO 1128 EVFTKP SEQ ID NO 1201 RQDNEI SEQ ID NO 1405 VRKSLK SEQ ID NO 1205 TKPLKA SEQ ID NO 1408 GNVNMW SEQ ID NO 1410 GGNFEG SEQ ID NO 1413 GKPRVR SEQ ID NO 1414 EILIEW SEQ ID NO 1416 IGYSFT SEQ ID NO 1417 PDEINT SEQ ID NO 1377 VFTKPQ SEQ ID NO 1153 DTSYVS SEQ ID NO 1334 YATKRQ SEQ ID NO 1350 VDGKPR SEQ ID NO 1353 NFEGSQ SEQ ID NO 1382 RALKYE SEQ ID NO 1373 LKYEVQ SEQ ID NO 1134 LKAPLT SEQ ID NO 1173 NVNMWD SEQ ID NO 1383 SRKAFV SEQ ID NO 1309 EGSQSL SEQ ID NO 1387 EQDSFG SEQ ID NO 1388 NTIYLG SEQ ID NO 1391 FVFPKE SEQ ID NO 1060 MSRKAF SEQ ID NO 1308 ESDTSY SEQ ID NO 1344 NEILIF SEQ ID NO 1399 PFSPNV SEQ ID NO 1402 DIGNVN SEQ ID NO 1404 NWRALK SEQ ID NO 1364 VFTKPQ SEQ ID NO 1153 IFWSKD SEQ ID NO 1181 SYVSLK SEQ ID NO 1407 SIFSYA SEQ ID NO 1409 SQSLVG SEQ ID NO 1411 LVGDIG SEQ ID NO 1327 FWSKDI  SEQ ID NO 1363 KKGYTV SEQ ID NO 1360 Elastin

Several candidate proteases may be responsible for the digestion of elastin in fibrotic tissue We have through a range of in vitro cleavages of pure native proteins determined that the enzymes listed in the following table cleaved elastin at least at the cleavage sites at each end of the following sequences or at the cleavage sites marked ‘.’ or where no ‘.’ is shown, at the ends of the sequences:

TABLE 24 Elastin fragments generated by specific proteases. Protease Sequence between cleavage sites Nos* MMP9 + 12 GVPGAIPGGVPG SEQ ID NO 1418 028-039 MMP9 + 12 AIPGGVPGGVFYPGAGLG SEQ ID NO 1419 032-049 MMP9 + 12 AIPGGVPGGVFYPGAGLGA SEQ ID NO 1420 032-050 MMP9 + 12 GVPGGVFYPGAGLGA SEQ ID NO 1421 036-050 MMP9 + 12 GVPGGVFYPGAGLGALG SEQ ID NO 1422 036-052 MMP9 + 12 VPGGVFYPGAGLGALGG SEQ ID NO 1423 037-053 MMP9 + 12 GVFYPGAGLGALGGGALGPGG SEQ ID NO 1424 040-060 MMP9 + 12 VFYPGAGLG SEQ ID NO 1425 041-049 MMP9 + 12 VFYPGAGLGA SEQ ID NO 1426 041-050 MMP9 + 12 VFYPGAGLGAL SEQ ID NO 1427 041-051 MMP9 + 12 VFYPGAGLGALG SEQ ID NO 1428 041-052 MMP9 + 12 VFYPGAGLGALGG SEQ ID NO 1429 041-053 MMP9 + 12 VFYPGAGLGALGGG SEQ ID NO 1430 041-054 MMP9 + 12 VFYPGAGLGALGGGAL SEQ ID NO 1431 041-056 MMP9 + 12 VFYPGAGLGALGGGALG SEQ ID NO 1432 041-057 MMP9 + 12 VFYPGAGLGALGGGALGPG SEQ ID NO 1433 041-059 MMP9 + 12 VFYPGAGLGALGGGALGPGG SEQ ID NO 1434 041-060 MMP9 + 12 VFYPGAGLGALGGGALGPGGKPLKPVPGG SEQ ID NO 1435 041-069 MMP9 + 12 LGALGGGALGPGGKPLKPVPGG SEQ ID NO 1436 048-069 MMP9 + 12 ALGGGALGPGGKPLKPVPGG SEQ ID NO 1437 050-069 MMP9 + 12 LGGGALGPGGKPLKPVPG SEQ ID NO 1438 051-068 MMP9 + 12 LGGGALGPGGKPLKPVPGG SEQ ID NO 1439 051-069 MMP9 + 12 GGALGPGGKPLKPVPGG SEQ ID NO 1440 053-069 MMP9 + 12 LGPGGKPLKPVPGG SEQ ID NO 1441 056-069 MMP9 + 12 GPGGKPLKPVPGG SEQ ID NO 1442 057-069 MMP9 + 12 PGGKPLKPVPGG SEQ ID NO 1443 058-069 MMP9 + 12 GKPLKPVPGG SEQ ID NO 1444 060-069 MMP9 + 12 PLKPVPGG SEQ ID NO 1445 062-069 MMP9 + 12 LKPVPGG SEQ ID NO  063-069 SEQ ID NO 1446 MMP9 + 12 GLAGAGLGAGLGAFP SEQ ID NO 1447 069-083 MMP9 + 12 GLAGAGLGAGLGAFPA SEQ ID NO 1448 069-084 MMP9 + 12 LAGAGLGAGLG SEQ ID NO 1449 070-080 MMP9 + 12 LAGAGLGAGLGAFP SEQ ID NO 1450 070-083 MMP9 + 12 LAGAGLGAGLGAFPA SEQ ID NO 1451 070-084 MMP9 + 12 LAGAGLGAGLGAFPAVT SEQ ID NO 1452 070-086 MMP9 + 12 LAGAGLGAGLGAFPAVTFPG SEQ ID NO 1453 070-089 MMP9 + 12 LAGAGLGAGLGAFPAVTFPGA SEQ ID NO 1454 070-090 MMP9 + 12 LAGAGLGAGLGAFPAVTFPGALVPGG SEQ ID NO 1455 070-095 MMP9 + 12 LAGAGLGAGLGAFPAVTFPGALVPGGVA SEQ ID NO 1456 070-097 MMP9 + 12 LAGAGLGAGLGAFPAVTFPGALVPGGVADAAAA SEQ ID NO 1457 070-102 MMP9 + 12 AGAGLGAGLGAFPAVTFPGALVPGG SEQ ID NO 1458 071-095 MMP9 + 12 GAGLGAGLGAFPA SEQ ID NO 1459 072-084 MMP9 + 12 GAGLGAGLGAFPAVTFPGA SEQ ID NO 1460 072-090 MMP9 + 12 AGLGAGLGAFPA SEQ ID NO 1461 073-084 MMP9 + 12 GLGAGLGAFPA SEQ ID NO 1462 074-084 MMP9 + 12 LGAGLGAFPA SEQ ID NO 1463 075-084 MMP9 + 12 LGAGLGAFPAVTFPGA SEQ ID NO 1464 075-090 MMP9 + 12 LGAGLGAFPAVTFPGALVPGG SEQ ID NO 1465 075-095 MMP9 + 12 LGAGLGAFPAVTFPGALVPGGVADAAAA SEQ ID NO 1466 075-102 MMP9 + 12 AGLGAFPAVTFPG SEQ ID NO 1467 077-089 MMP9 + 12 LGAFPAVTFPGA SEQ ID NO 1468 079-090 MMP9 + 12 LGAFPAVTFPGALVPGGVA SEQ ID NO 1469 079-097 MMP9 + 12 LGAFPAVTFPGALVPGGVADAAAA SEQ ID NO 1470 079-102 MMP9 + 12 AFPAVTFPGALVPGG SEQ ID NO 1471 081-095 MMP9 + 12 AVTFPGALVPGG SEQ ID NO 1472 084-095 MMP9 + 12 AVTFPGALVPGGVADAAAA SEQ ID NO 1473 084-102 MMP9 + 12 VTFPGALVPGG SEQ ID NO 1474 085-095 MMP9 + 12 VTFPGALVPGGVADAAAA SEQ ID NO 1475 085-102 MMP9 + 12 LVPGGVADAAAA SEQ ID NO 1476 091-102 MMP9 + 12 LVPGGVADAAAAYK SEQ ID NO 1477 091-104 MMP9 + 12 VADAAAAYK SEQ ID NO 1478 096-104 MMP9 + 12 KAAKAGA SEQ ID NO 1479 104-110 MMP9 + 12 LGVSAGAVVPQPGA SEQ ID NO 1480 121-134 MMP9 + 12 VPGVGLPGVYPGGVLPGAR SEQ ID NO 1481 141-159 MMP9 + 12 PGVGLPGVYPGGVLPGAR SEQ ID NO 1482 142-159 MMP9 + 12 GLPGVYPGGVLPGAR SEQ ID NO 1483 145-159 MMP9 + 12 PGVYPGGVLPGAR SEQ ID NO 1484 147-159 MMP9 + 12 ARFPGVG SEQ ID NO 1485 158-164 MMP9 + 12 ARFPGVGVLPG SEQ ID NO 1486 158-168 MMP9 + 12 RFPGVGVLPGVPTGAG SEQ ID NO 1487 159-174 MMP9 + 12 FPGVGVLPGVPTG SEQ ID NO 1488 160-172 MMP9 + 12 FPGVGVLPGVPTGA SEQ ID NO 1489 160-173 MMP9 + 12 FPGVGVLPGVPTGAGV SEQ ID NO 1490 160-175 MMP9 + 12 FPGVGVLPGVPTGAGVKPK SEQ ID NO 1491 160-178 MMP9 + 12 KPKAPGV SEQ ID NO 1492 176-182 MMP9 + 12 PKAPGV SEQ ID NO 1493 177-182 MMP9 + 12 GAFAGIPGVGPFG SEQ ID NO 1494 184-196 MMP9 + 12 VGPFGGPQPGVPLGYP SEQ ID NO 1495 192-207 MMP9 + 12 GPQPGVPLGYP SEQ ID NO 1496 197-207 MMP9 + 12 PQPGVPLGYP SEQ ID NO 1497 198-207 MMP9 + 12 PGVPLGYP SEQ ID NO 1498 200-207 MMP9 + 12 GYPIKAPK SEQ ID NO 1499 205-212 MMP9 + 12 PKLPGGY SEQ ID NO 1500 211-217 MMP9 + 12 YTTGKLPYGYGPG SEQ ID NO 1501 221-233 MMP9 + 12 YTTGKLPYGYGPGGVAGAAGK SEQ ID NO 1502 221-241 MMP9 + 12 TTGKLPYGYG SEQ ID NO 1503 222-231 MMP9 + 12 TTGKLPYGYGPGGVAGAAGK SEQ ID NO 1504 222-241 MMP9 + 12 LPYGYGPGGVAGAAGK SEQ ID NO 1505 226-241 MMP9 + 12 GYGPGGVAGAAGK SEQ ID NO 1506 229-241 MMP9 + 12 YGPGGVAGAAGK SEQ ID NO 1507 230-241 MMP9 + 12 AGYPTGTGVGPQAAAAAAAK SEQ ID NO 1508 242-261 MMP9 + 12 TGVGPQAAAAAAAK SEQ ID NO 1509 248-261 MMP9 + 12 PQAAAAAAAK SEQ ID NO 1510 252-261 MMP9 + 12 FGAGAAGVLPGVGGAGVPGVPGAIPGIGG SEQ ID NO 1511 266-294 MMP9 + 12 FGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAA SEQ ID NO 1512 266-303 MMP9 + 12 GVLPGVGGAGVPGVPG SEQ ID NO 1513 272-287 MMP9 + 12 VLPGVGGAGVPGVPGAIPGIGG SEQ ID NO 1514 273-294 MMP9 + 12 VLPGVGGAGVPGVPGAIPGIGGIAGVGTPA SEQ ID NO 1515 273-302 MMP9 + 12 VLPGVGGAGVPGVPGAIPGIGGIAGVGTPAA SEQ ID NO 1516 273-303 MMP9 + 12 GAG VPG VPGAIPG SEQ ID NO 1517 279-291 MMP9 + 12 GAGVPGVPGAIPGIGGIAGVG SEQ ID NO 1518 279-299 MMP9 + 12 AGVPGVPGAIPGIG SEQ ID NO 1519 280-293 MMP9 + 12 AGVPGVPGAIPGIGG SEQ ID NO 1520 280-294 MMP9 + 12 AGVPGVPGAIPGIGGIAG SEQ ID NO 1521 280-297 MMP9 + 12 AGVPGVPGAIPGIGGIAGVGTPA SEQ ID NO 1522 280-302 MMP9 + 12 GVPGVPGAIPGIGG SEQ ID NO 1523 281-294 MMP9 + 12 GVPGVPGAIPGIGGIA SEQ ID NO 1524 281-296 MMP9 + 12 GVPGVPGAIPGIGGIAGVG SEQ ID NO 1525 281-299 MMP9 + 12 VPGVPGAIPGIGG SEQ ID NO 1526 282-294 MMP9 + 12 GVPGAIPGIGGIAGVGTPA SEQ ID NO 1527 284-302 MMP9 + 12 VPGAIPGIGGIAGVG SEQ ID NO 1528 285-299 MMP9 + 12 VPGAIPGIGGIAGVGTPA SEQ ID NO 1529 285-302 MMP9 + 12 VPGAIPGIGGIAGVGTPAAA SEQ ID NO 1530 285-304 MMP9 + 12 VPGAIPGIGGIAGVGTPAAAAAAAAAAK SEQ ID NO 1531 285-312 MMP9 + 12 AIPGIGGIAGVG SEQ ID NO 1532 288-299 MMP9 + 12 AIPGIGGIAGVGTPA SEQ ID NO 1533 288-302 MMP9 + 12 AIPGIGGIAGVGTPAA SEQ ID NO 1534 288-303 MMP9 + 12 AIPGIGGIAGVGTPAAA SEQ ID NO 1535 288-304 MMP9 + 12 AIPGIGGIAGVGTPAAAAAA SEQ ID NO 1536 288-307 MMP9 + 12 AIPGIGGIAGVGTPAAAAAAAAAAK SEQ ID NO 1537 288-312 MMP9 + 12 IPGIGGIAGVGTPAAA SEQ ID NO 1538 289-304 MMP9 + 12 IGGIAGVGTPAAAA SEQ ID NO 1539 292-305 MMP9 + 12 GIAGVGTPAAAA SEQ ID NO 154O 294-305 MMP9 + 12 GIAGVGTPAAAAAAAA SEQ ID NO 1541 294-309 MMP9 + 12 GIAGVGTPAAAAAAAAAAK SEQ ID NO 1542 294-312 MMP9 + 12 IAGVGTPAAAAAAAA SEQ ID NO 1543 295-309 MMP9 + 12 IAGVGTPAAAAAAAAA SEQ ID NO 1544 295-310 MMP9 + 12 IAGVGTPAAAAAAAAAAK SEQ ID NO 1545 295-312 MMP9 + 12 TPAAAAAAAAAAK SEQ ID NO 1546 300-312 MMP9 + 12 PAAAAAAAAAAK SEQ ID NO 1547 301-312 MMP9 + 12 AAAAAAAAAAK SEQ ID NO 1548 302-312 MMP9 + 12 AAAAAAAAAK SEQ ID NO 1549 303-312 MMP9 + 12 AAAAAAAAK SEQ ID NO 1550 304-312 MMP9 + 12 AAAAAAAAKA SEQ ID NO 1551 304-313 MMP9 + 12 AAAAAAAK SEQ ID NO 1552 305-312 MMP9 + 12 LVPGGPGFGPGVVGVPGA SEQ ID NO 1553 322-339 MMP9 + 12 GPGFGPGVVGVPG SEQ ID NO 1554 326-338 MMP9 + 12 GPGFGPGVVGVPGAGVPGVG SEQ ID NO 1555 326-345 MMP9 + 12 GPGFGPGVVGVPGAGVPGVGVPGAGIPVVPG SEQ ID NO 1556 326-356 MMP9 + 12 PGFGPGVVGVPG SEQ ID NO 1557 327-338 MMP9 + 12 PGFGPGVVGVPGA SEQ ID NO 1558 327-339 MMP9 + 12 PGFGPGVVGVPGAG SEQ ID NO 1559 327-340 MMP9 + 12 PGVVGVPGAGVPG SEQ ID NO 1560 331-343 MMP9 + 12 PGVVGVPGAGVPGVGVPG SEQ ID NO 1561 331-348 MMP9 + 12 PGVVGVPGAGVPGVGVPGAGIPVVPGA SEQ ID NO 1562 331-357 MMP9 + 12 VVGVPGAGVPGVGVPGA SEQ ID NO 1563 333-349 MMP9 + 12 VGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPEA SEQ ID NO 1564 334-372 MMP9 + 12 AGVPGVGVPGAGIPVVPG SEQ ID NO 1565 339-356 MMP9 + 12 GVPGVGVPGAGIPVVPG SEQ ID NO 1566 340-356 MMP9 + 12 GVPGVGVPGAGIPVVPGA SEQ ID NO 1567 340-357 MMP9 + 12 VPGVGVPGAGIPVVPG SEQ ID NO 1568 341-356 MMP9 + 12 VGVPGAGIPVVPG SEQ ID NO 1569 344-356 MMP9 + 12 VGVPGAGIPVVPGAGIPG SEQ ID NO 1570 344-361 MMP9 + 12 VPGAGIPVVPG SEQ ID NO 1571 346-356 MMP9 + 12 AGIPVVPGAGIPG SEQ ID NO 1572 349-361 MMP9 + 12 AGIPVVPGAGIPGAAVPGVVSPEAAAK SEQ ID NO 1573 349-375 MMP9 + 12 GIPVVPGAGIPG SEQ ID NO 1574 350-361 MMP9 + 12 IPGAAVPGVVSPEAAAK SEQ ID NO 1575 359-375 MMP9 + 12 GAAVPGVVSPEAAAK SEQ ID NO 1576 361-375 MMP9 + 12 AVPGVVSPEAAAK SEQ ID NO 1577 363-375 MMP9 + 12 VPGVVSPEAAAK SEQ ID NO 1578 364-375 MMP9 + 12 YGARPGVG SEQ ID NO 1579 383-390 MMP9 + 12 YGARPGVGVG SEQ ID NO 1580 383-392 MMP9 + 12 YGARPGVGVGGIPT SEQ ID NO 1581 383-396 MMP9 + 12 YGARPGVGVGGIPTY SEQ ID NO 1582 383-397 MMP9 + 12 YGARPGVGVGGIPTYG SEQ ID NO 1583 383-398 MMP9 + 12 YGARPGVGVGGIPTYGVG SEQ ID NO 1584 383-400 MMP9 + 12 YGARPGVGVGGIPTYGVGA SEQ ID NO 1585 383-401 MMP9 + 12 YGARPGVGVGGIPTYGVGAG SEQ ID NO 1586 383-402 MMP9 + 12 GARPGVGV SEQ ID NO 1587 384-391 MMP9 + 12 GARPGVGVGG SEQ ID NO 1588 384-393 MMP9 + 12 GARPGVGVGGIP SEQ ID NO 1589 384-395 MMP9 + 12 GARPGVGVGGIPTY SEQ ID NO 1590 384-397 MMP9 + 12 GARPGVGVGGIPTYGV SEQ ID NO 1591 384-399 MMP9 + 12 GARPGVGVGGIPTYGVG SEQ ID NO 1592 384-400 MMP9 + 12 GARPGVGVGGIPTYGVGAGGF SEQ ID NO 1593 384-404 MMP9 + 12 GARPGVGVGGIPTYGVGAGGFPGF SEQ ID NO 1594 384-407 MMP9 + 12 GARPGVGVGGIPTYGVGAGGFPGFG SEQ ID NO 1595 384-408 MMP9 + 12 GARPGVGVGGIPTYGVGAGGFPGFGVGVG SEQ ID NO 1596 384-412 MMP9 + 12 ARPGVGVGG SEQ ID NO 1597 385-393 MMP9 + 12 ARPGVGVGGIP SEQ ID NO 1598 385-395 MMP9 + 12 ARPGVGVGGIPTY SEQ ID NO 1599 385-397 MMP9 + 12 ARPGVGVGGIPTYGVGA SEQ ID NO 1600 385-401 MMP9 + 12 ARPGVGVGGIPTYGVGAGG SEQ ID NO 1601 385-403 MMP9 + 12 ARPGVGVGGIPTYGVGAGGFPG SEQ ID NO 1602 385-406 MMP9 + 12 ARPGVGVGGIPTYGVGAGGFPGF SEQ ID NO 1603 385-407 MMP9 + 12 RPGVGVG SEQ ID NO 1604 386-392 MMP9 + 12 RPGVGVGG SEQ ID NO 1605 386-393 MMP9 + 12 PGVGVGGIPTY SEQ ID NO 1606 387-397 MMP9 + 12 PGVGVGGIPTYG SEQ ID NO 1607 387-398 MMP9 + 12 PGVGVGGIPTYGVGAG SEQ ID NO 1608 387-412 MMP9 + 12 VGGIPTYGVGAG SEQ ID NO 1609 391-402 MMP9 + 12 GVGAGGFPGFGVGVGGIPGVA SEQ ID NO 1610 398-418 MMP9 + 12 VGAGGFPGFGVGVG SEQ ID NO 1611 399-412 MMP9 + 12 VGVGGIPGVAGVPSVGGVPGVGGVPGVGISPEA SEQ ID NO 1612 409-441 MMP9 + 12 VAGVPSVGGVPGVGGVPG SEQ ID NO 1613 417-434 MMP9 + 12 VAGVPSVGGVPGVGGVPGVGISPEA SEQ ID NO 1614 417-441 MMP9 + 12 SVGGVPGVGGVPGVGISPEA SEQ ID NO 1615 422-441 MMP9 + 12 VGGVPGVGGVPGVGISPEA SEQ ID NO 1616 423-441 MMP9 + 12 GVPGVGGVPGVGIS SEQ ID NO 1617 425-438 MMP9 + 12 GVPGVGGVPGVGIS PEA SEQ ID NO 1618 425-441 MMP9 + 12 GVPGVGGVPGVGISPEAQA SEQ ID NO 1619 425-443 MMP9 + 12 GVPGVGISPEAQAAAAAK SEQ ID NO 1620 431-448 MMP9 + 12 GVGTPAAAAAK SEQ ID NO 1621 482-492 MMP9 + 12 TPAAAAAK SEQ ID NO 1622 485-492 MMP9 + 12 FGLVPGVGVAPGVG SEQ ID NO 1623 500-513 MMP9 + 12 FGLVPGVGVAPGVGVAPG SEQ ID NO 1624 500-517 MMP9 + 12 FGLVPGVGVAPGVGVAPGVGVAPG SEQ ID NO 1625 500-523 MMP9 + 12 FGLVPGVGVAPGVGVAPGVGVAPGVG SEQ ID NO 1626 500-525 MMP9 + 12 FGLVPGVGVAPGVGVAPGVGVAPGVGLAPG SEQ ID NO 1627 500-529 MMP9 + 12 FGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPG SEQ ID NO 1628 500-535 MMP9 + 12 FGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGV SEQ ID NO 1629 500-536 MMP9 + 12 FGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGV SEQ ID NO 1630 500-541 GVAPG MMP9 + 12 GLVPGVGVAPG SEQ ID NO 1631 501-511 MMP9 + 12 GLVPGVGVAPGV SEQ ID NO 1632 501-512 MMP9 + 12 GLVPGVGVAPGVGVA SEQ ID NO 1633 501-515 MMP9 + 12 GLVPGVGVAPGVGVAP SEQ ID NO 1634 501-516 MMP9 + 12 GLVPGVGVAPGVGVAPG SEQ ID NO 1635 501-517 MMP9 + 12 GLVPGVGVAPGVGVAPGVG SEQ ID NO 1636 501-519 MMP9 + 12 GLVPGVGVAPGVGVAPGVGVAPG SEQ ID NO 1637 501-523 MMP9 + 12 GLVPGVGVAPGVGVAPGVGVAPGVGL SEQ ID NO 1638 501-524 MMP9 + 12 GLVPGVGVAPGVGVAPGVGVAPGVGLA SEQ ID NO 1639 501-525 MMP9 + 12 GLVPGVGVAPGVGVAPGVGVAPGVGLAPG SEQ ID NO 1640 501-527 MMP9 + 12 GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVG SEQ ID NO 1641 501-529 MMP9 + 12 GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVA SEQ ID NO 1642 501-531 MMP9 + 12 GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPG SEQ ID NO 1643 501-533 MMP9 + 12 LVPGVGVAPGVG SEQ ID NO 1644 502-513 MMP9 + 12 LVPGVGVAPGVGVAPG SEQ ID NO 1645 502-517 MMP9 + 12 LVPGVGVAPGVGVAPGVG SEQ ID NO 1646 502-519 MMP9 + 12 LVPGVGVAPGVGVAPGVGVAPGVG SEQ ID NO 1647 502-525 MMP9 + 12 LVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVG SEQ ID NO 1648 502-537 MMP9 + 12 PGVGVAPGVGVAPG SEQ ID NO 1649 504-517 MMP9 + 12 VGVAPGVGVAPGVGV SEQ ID NO 1650 506-520 MMP9 + 12 VGVAPGVGVAPGVGVAPGVG SEQ ID NO 1651 506-525 MMP9 + 12 VGVAPGVGVAPGVGVAPGVGLAPGVGVAPG SEQ ID NO 1652 506-535 MMP9 + 12 VAPGVGVAPGVGVAPG SEQ ID NO 1653 508-523 MMP9 + 12 VAPGVGVAPGVGVAPGVG SEQ ID NO 1654 508-525 MMP9 + 12 VAPGVGVAPGVGVAPGVGLAPGVG SEQ ID NO 1655 508-531 MMP9 + 12 VAPGVGVAPGVGVAPGVGLAPGVGVAPG SEQ ID NO 1656 508-535 MMP9 + 12 VGVAPGVGVAPGVGLA SEQ ID NO 1657 512-527 MMP9 + 12 VGVAPGVGVAPGVGLAPGVGVAPG SEQ ID NO 1658 512-535 MMP9 + 12 VGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1659 512-552 MMP9 + 12 VAPGVGVAPGVGLAPGVGVAPGVG SEQ ID NO 1660 514-537 MMP9 + 12 VAPGVGVAPGVGLAPGVGVAPGVGVA SEQ ID NO 1661 514-539 MMP9 + 12 VAPGVGVAPGVGLAPGVGVAPGVGVAPG SEQ ID NO 1662 514-541 MMP9 + 12 VAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGP SEQ ID NO 1663 514-550 MMP9 + 12 VAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1664 514-552 MMP9 + 12 PGVGVAPGVGLAPG SEQ ID NO 1665 516-529 MMP9 + 12 PGVGVAPGVGLAPGVGVAP SEQ ID NO 1666 516-534 MMP9 + 12 PGVGVAPGVGLAPGVGVAPGVG SEQ ID NO 1667 516-537 MMP9 + 12 VGVAPGVGLAPGVGVA SEQ ID NO 1668 518-533 MMP9 + 12 VGVAPGVGLAPGVGVAP SEQ ID NO 1669 518-534 MMP9 + 12 VGVAPGVGLAPGVGVAPGVGVAPG SEQ ID NO 1670 518-541 MMP9 + 12 VGVAPGVGLAPGVGVAPGVGVAPGVG SEQ ID NO 1671 518-543 MMP9 + 12 VGVAPGVGLAPGVGVAPGVGVAPGVGVAPG SEQ ID NO 1672 518-547 MMP9 + 12 VGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1673 518-552 MMP9 + 12 GVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1674 519-552 MMP9 + 12 VAPGVGLAPGVGVA SEQ ID NO 1675 520-533 MMP9 + 12 VAPGVGLAPGVGVAPG SEQ ID NO 1676 520-535 MMP9 + 12 VAPGVGLAPGVGVAPGVG SEQ ID NO 1677 520-537 MMP9 + 12 VAPGVGLAPGVGVAPGVGVA SEQ ID NO 1678 520-539 MMP9 + 12 VAPGVGLAPGVGVAPGVGVAPG SEQ ID NO 1679 520-541 MMP9 + 12 VAPGVGLAPGVGVAPGVGVAPGVGVA SEQ ID NO 1680 520-545 MMP9 + 12 VAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1681 520-552 MMP9 + 12 PGVGLAPGVGVAPG SEQ ID NO 1682 522-535 MMP9 + 12 GVGLAPGVGVAPGVGVAPG SEQ ID NO 1683 523-541 MMP9 + 12 VGLAPGVGVAPGVG SEQ ID NO 1684 524-537 MMP9 + 12 VGLAPGVGVAPGVGVAPG SEQ ID NO 1685 524-541 MMP9 + 12 VGLAPGVGVAPGVGVAPGVGVAPGIG SEQ ID NO 1686 524-549 MMP9 + 12 VGLAPGVGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1687 524-552 MMP9 + 12 VGLAPGVGVAPGVGVAPGVGVAPGIGPG SEQ ID NO 1688 524-553 MMP9 + 12 VGLAPGVGVAPGVGVAPGVGVAPGIGPGGVAAA SEQ ID NO 1689 524-556 MMP9 + 12 LAPGVGVAPGVGVAPGVG SEQ ID NO 1690 526-543 MMP9 + 12 LAPGVGVAPGVGVAPGVGVA SEQ ID NO 1691 526-545 MMP9 + 12 LAPGVGVAPGVGVAPGVGVAPGIGP SEQ ID NO 1692 526-550 MMP9 + 12 LAPGVGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1693 526-552 MMP9 + 12 GVGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1694 529-552 MMP9 + 12 VGVAPGVGVAPGVGVA SEQ ID NO 1695 530-545 MMP9 + 12 VGVAPGVGVAPGVGVAPG SEQ ID NO 1696 530-547 MMP9 + 12 VGVAPGVGVAPGVGVAPGIGPG SEQ ID NO 1697 530-551 MMP9 + 12 VGVAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1698 530-552 MMP9 + 12 VGVAPGVGVAPGVGVAPGIGPGGVAAA SEQ ID NO 1699 530-556 MMP9 + 12 VAPGVGVAPGVGVAP SEQ ID NO 1700 532-546 MMP9 + 12 VAPGVGVAPGVGVAPGIG SEQ ID NO 1701 532-549 MMP9 + 12 VAPGVGVAPGVGVAPGIGPGG SEQ ID NO 1702 532-552 MMP9 + 12 PGVGVAPGVGVAPGIGPG SEQ ID NO 1703 534-551 MMP9 + 12 PGVGVAPGVGVAPGIGPGG SEQ ID NO 1704 534-552 MMP9 + 12 VGVAPGVGVAPGIGPGG SEQ ID NO 1705 536-552 MMP9 + 12 VGVAPGVGVAPGIGPGGVAA SEQ ID NO 1706 536-555 MMP9 + 12 VAPGVGVAPGIGPG SEQ ID NO 1707 538-551 MMP9 + 12 PGVGVAPGIGPG SEQ ID NO 1708 540-551 MMP9 + 12 VGVAPGIGPGGVAA SEQ ID NO 1709 542-555 MMP9 + 12 PGGVAAAAK SEQ ID NO 1710 550-558 MMP9 + 12 LRAAAGL SEQ ID NO 1711 569-575 MMP9 + 12 LRAAAGLG SEQ ID NO 1712 569-576 MMP9 + 12 LRAAAGLGA SEQ ID NO 1713 569-577 MMP9 + 12 AAAGLGAGIPGLGVG SEQ ID NO 1714 571-585 MMP9 + 12 AAAGLGAGIPGLGVGVG SEQ ID NO 1715 571-587 MMP9 + 12 LGAGIPGLGVG SEQ ID NO 1716 575-585 MMP9 + 12 LGAGIPGLGVGVG SEQ ID NO 1717 575-587 MMP9 + 12 LGAGIPGLGVGVGVPGLGVG SEQ ID NO 1718 575-594 MMP9 + 12 LGAGIPGLGVGVGVPG SEQ ID NO 1719 575-590 MMP9 + 12 LGAGIPGLGVGVGVPGLGVGA SEQ ID NO 1720 575-595 MMP9 + 12 LGAGIPGLGVGVGVPGL SEQ ID NO 1721 575-591 MMP9 + 12 LGAGIPGLGVGVGVPGLG SEQ ID NO 1722 575-592 MMP9 + 12 LGAGIPGLGVGVGVPGLGVGAGVPG SEQ ID NO 1723 575-599 MMP9 + 12 LGAGIPGLGVGVGVPGLGVGAGVPGLG SEQ ID NO 1724 575-601 MMP9 + 12 LGAGIPGLGVGVGVPGLGVGAGVPGLGVG SEQ ID NO 1725 575-603 MMP9 + 12 LGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFG SEQ ID NO 1726 575-610 MMP9 + 12 GAGIPGLGVGVGVPGLG SEQ ID NO 1727 576-592 MMP9 + 12 AGIPGLGVGVGVPG SEQ ID NO 1728 577-590 MMP9 + 12 GIPGLGVGVGVPGLGVGA SEQ ID NO 1729 578-595 MMP9 + 12 LGVGVGVPGLGVGA SEQ ID NO 1730 582-595 MMP9 + 12 VGVPGLGVGAGVPG SEQ ID NO 1731 586-599 MMP9 + 12 VGVPGLGVGAGVPGL SEQ ID NO 1732 586-600 MMP9 + 12 VGVPGLGVGAGVPGLG SEQ ID NO 1733 586-601 MMP9 + 12 VGVPGLGVGAGVPGLGVG SEQ ID NO 1734 586-603 MMP9 + 12 VGVPGLGVGAGVPGLGVGA SEQ ID NO 1735 586-604 MMP9 + 12 VGAGVPGLGVGAGVPGFG SEQ ID NO 1736 593-610 MMP9 + 12 PGALAAAK SEQ ID NO 1737 646-653 MMP9 + 12 AKYGAAVPGVLGGLGA SEQ ID NO 1738 655-670 MMP9 + 12 YGAAVPGVLGG SEQ ID NO 1739 657-667 MMP9 + 12 YGAAVPGVLGGLG SEQ ID NO 1740 657-669 MMP9 + 12 YGAAVPGVLGGLGA SEQ ID NO 1741 657-670 MMP9 + 12 YGAAVPGVLGGLGALG SEQ ID NO 1742 657-672 MMP9 + 12 YGAAVPGVLGGLGALGGVGIPGG SEQ ID NO 1743 657-679 MMP9 + 12 YGAAVPGVLGGLGALGGVGIPGGVVGAGPAA SEQ ID NO 1744 657-687 MMP9 + 12 GAAVPGVLGGLG SEQ ID NO 1745 658-669 MMP9 + 12 GAAVPGVLGGLGALGGVGIPGG SEQ ID NO 1746 658-679 MMP9 + 12 AVPGVLGGLGA SEQ ID NO 1747 660-670 MMP9 + 12 AVPGVLGGLGALGGVGIPGG SEQ ID NO 1748 660-679 MMP9 + 12 VLGGLGALGGVGIPGG SEQ ID NO 1749 664-679 MMP9 + 12 GGLGALGGVGIPGGVVGAGPA SEQ ID NO 1750 666-686 MMP9 + 12 GGLGALGGVGIPGGVVGAGPAAA SEQ ID NO 1751 666-688 MMP9 + 12 LGALGGVGIPGG SEQ ID NO 1752 668-379 MMP9 + 12 LGALGGVGIPGGVVGAGPA SEQ ID NO 1753 668-686 MMP9 + 12 LGALGGVGIPGGVVGAGPAA SEQ ID NO 1754 668-687 MMP9 + 12 LGALGGVGIPGGVVGAGPAAA SEQ ID NO 1755 668-688 MMP9 + 12 LGALGGVGIPGGVVGAGPAAAA SEQ ID NO 1756 668-689 MMP9 + 12 ALGGVGIPGGVVGAGPAA SEQ ID NO 1757 670-687 MMP9 + 12 ALGGVGIPGGVVGAGPAAA SEQ ID NO 1758 670-688 MMP9 + 12 LGGVGIPGGV SEQ ID NO 1759 671-680 MMP9 + 12 LGGVGIPGGVVGAGPA SEQ ID NO 1760 671-686 MMP9 + 12 LGGVGIPGGVVGAGPAAA SEQ ID NO 1761 671-688 MMP9 + 12 LGGVGIPGGVVGAGPAAAAA SEQ ID NO 1762 671-690 MMP9 + 12 LGGVGIPGGVVGAGPAAAAAAAK SEQ ID NO 1763 671-693 MMP9 + 12 GVGIPGGVVGAGPAAAA SEQ ID NO 1764 673-689 MMP9 + 12 GVGIPGGVVGAGPAAAAAAAK SEQ ID NO 1765 673-693 MMP9 + 12 VGIPGGVVGAGPAAA SEQ ID NO 1766 674-688 MMP9 + 12 VGIPGGVVGAGPAAAAAAAK SEQ ID NO 1767 674-693 MMP9 + 12 IPGGVVGAGPAAAA SEQ ID NO 1768 676-689 MMP9 + 12 VVGAGPAAAAAAAK SEQ ID NO 1769 680-693 MMP9 + 12 VGAGPAAAAAAAK SEQ ID NO 1770 681-693 MMP9 + 12 AGPAAAAAAAK SEQ ID NO 1771 683-693 MMP9 + 12 GPAAAAAAAK SEQ ID NO 1772 684-693 MMP9 + 12 PAAAAAAAK SEQ ID NO 1773 685-693 MMP9 + 12 FGLVGAAGLGGLGVGGLGVPGVGG SEQ ID NO 1774 701-724 MMP9 + 12 GLVGAAGLGGLG SEQ ID NO 1775 702-713 MMP9 + 12 GLVGAAGLGGLGVGG SEQ ID NO 1776 702-716 MMP9 + 12 GLVGAAGLGGLGVGGLGVPGVG SEQ ID NO 1777 702-723 MMP9 + 12 GLVGAAGLGGLGVGGLGVPGVGG SEQ ID NO 1778 702-724 MMP9 + 12 LVGAAGLGGLGVG SEQ ID NO 1779 703-715 MMP9 + 12 LVGAAGLGGLGVGG SEQ ID NO 1780 703-716 MMP9 + 12 LVGAAGLGGLGVGGL SEQ ID NO 1781 703-717 MMP9 + 12 LVGAAGLGGLGVGGLGVPGVGGLG SEQ ID NO 1782 703-726 MMP9 + 12 LVGAAGLGGLGVGGLGVPGVGGLGGIPPAAA SEQ ID NO 1783 703-733 MMP9 + 12 VGAAGLGGLGVGG SEQ ID NO 1784 704-716 MMP9 + 12 LGGLGVGGLGVPG SEQ ID NO 1785 709-721 MMP9 + 12 LGGLGVGGLGVPGVG SEQ ID NO 1786 709-723 MMP9 + 12 LGGLGVGGLGVPGVGGL SEQ ID NO 1787 709-725 MMP9 + 12 LGGLGVGGLGVPGVGGLG SEQ ID NO 1788 709-726 MMP9 + 12 LGVGGLGVPGVGGLG SEQ ID NO 1789 712-726 MMP9 + 12 GLGVPGVGGLGGIPPAAAAK SEQ ID NO 1790 716-735 MMP9 + 12 LGGIPPAAAAK SEQ ID NO 1791 725-735 MMP9 + 12 LGGVLGGAGQFPL SEQ ID NO 1792 744-756 MMP9 + 12 LGGVLGGAGQFPLGGVAAR SEQ ID NO 1793 744-762 MMP9 + 12 LGGVLGGAGQFPLGGVAARPG SEQ ID NO 1794 744-764 MMP9 + 12 LGGVLGGAGQFPLGGVAARPGFG SEQ ID NO 1795 744-766 MMP9 + 12 GGVLGGAGQFPLGGVAARPG SEQ ID NO 1796 745-764 MMP9 + 12 GAGQFPLGGVAAR SEQ ID NO 1797 750-762 MMP9 + 12 GAGQFPLGGVAARPGFG SEQ ID NO 1798 750-766 MMP9 + 12 AGQFPLGGVAARPGFG SEQ ID NO 1799 751-766 MMP9 + 12 FPLGGVAARPG SEQ ID NO 1800 754-764 MMP9 + 12 PLGGVAAR SEQ ID NO 1801 755-762 MMP9 + 12 PLGGVAARPG SEQ ID NO 1802 755-764 MMP9 + 12 PLGGVAARPGFG SEQ ID NO 1803 755-766 MMP9 + 12 PLGGVAARPGFGL SEQ ID NO 1804 755-767 MMP9 + 12 PLGGVAARPGFGLSPIFPG SEQ ID NO 1805 755-773 MMP9 + 12 LGGVAAR SEQ ID NO 1806 756-762 MMP9 + 12 LGGVAARP SEQ ID NO 1807 756-763 MMP9 + 12 LGGVAARPG SEQ ID NO 1808 756-764 MMP9 + 12 LGGVAARPGF SEQ ID NO 1809 756-765 MMP9 + 12 LGGVAARPGFG SEQ ID NO 1810 756-766 MMP9 + 12 LGGVAARPGFGL SEQ ID NO 1811 756-767 MMP9 + 12 LGGVAARPGFGLSP SEQ ID NO 1812 756-769 MMP9 + 12 LGGVAARPGFGLSPIFPG SEQ ID NO 1813 756-773 MMP9 + 12 LGGVAARPGFGLSPIFPGG SEQ ID NO 1814 756-774 MMP9 + 12 LGGVAARPGFGLSPIFPGGA SEQ ID NO 1815 756-775 MMP9 + 12 GGVAARPGFG SEQ ID NO 1816 757-766 MMP9 + 12 GGVAARPGFGL SEQ ID NO 1817 757-767 MMP9 + 12 GGVAARPGFGLSPIFPGGA SEQ ID NO 1818 757-775 MMP9 + 12 GVAARPGFGLSPIF SEQ ID NO 1819 758-771 MMP9 + 12 GVAARPGFGLSPIFP SEQ ID NO 1820 758-772 MMP9 + 12 VAARPGFG SEQ ID NO 1821 759-766 MMP9 + 12 VAARPGFGLSPIFP SEQ ID NO 1822 759-772 MMP9 + 12 VAARPGFGLSPIFPG SEQ ID NO 1823 759-773 MMP9 + 12 RPGFGLSPIFPG SEQ ID NO 1824 762-773 MMP9 + 12 PGFGLSPIFPGG SEQ ID NO 1825 763-774 MMP9 + 12 PGFGLSPIFPGGA SEQ ID NO 1826 763-775 ADAMTS-1 P.GVGLPGVYPGGVLPGAR.F SEQ ID NO 1827 143-159 ADAMTS-1 G.VGLPGVYPGGVLPGAR.F SEQ ID NO 1828 144-159 ADAMTS-1 G.LPGVYPGGVLPGAR.F SEQ ID NO 1829 146-159 ADAMTS-1 P.GVYPGGVLPGAR.F SEQ ID NO 1830 148-159 ADAMTS-1 K.AGYPTGTGVGPQAAAAAAAK.A SEQ ID NO 1831 242-261 ADAMTS-1 G.GPGFGPGVVGVPGAGVPGVGVPGA.G SEQ ID NO 1832 326-349 ADAMTS-1 G.FGPGVVGVPGAGVPGVGVPG.A SEQ ID NO 1833 329-348 ADAMTS-1 F.GPGVVGVPGAGVPGVGVPG.A SEQ ID NO 1834 330-348 ADAMTS-1 G.VPGVGVPGAGIPVVPG.A SEQ ID NO 1835 341-356 ADAMTS-1 G.ARPGVGVGGIPTYGVG.A SEQ ID NO 1836 385-400 ADAMTS-1 G.ARPGVGVGGIPTYGVGAGG.F SEQ ID NO 1837 385-403 ADAMTS-1 A.RPGVGVGGIPTYGVGAG.G SEQ ID NO 1838 386-402 ADAMTS-1 G.GVPGVGGVPGVGISPEAQAAAA.A SEQ ID NO 1839 425-446 ADAMTS-1 G.VPGVGISPEAQAAAAAK.A SEQ ID NO 1840 432-448 ADAMTS-1 G.VGISPEAQAAAAAK.A SEQ ID NO 1841 435-448 ADAMTS-1 V.PGVGVAPGVGVAPGVGVAPGVGL.A SEQ ID NO 1842 504-526 ADAMTS-1 G.VAPGVGVAPGVGVAPGVGLAPGVGVAPG.V SEQ ID NO 1843 508-535 ADAMTS-1 G.VGVAPGVGVAPGVGLAPGVG.V SEQ ID NO 1844 512-531 ADAMTS-1 G.VGVAPGVGVAPGVGLAPGVGVAPGVG.V SEQ ID NO 1845 512-537 ADAMTS-1 A.PGVGVAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1846 528-551 ADAMTS-1 G.VAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1847 532-551 ADAMTS-1 G.AAVPGVLGGLGALGGVGIPG.G SEQ ID NO 1848 659-678 ADAMTS-1 G.AAGLGGLGVGGLGVPGVGGLG.G SEQ ID NO 1849 706-726 ADAMTS-4 P.GVGLPGVYPGGVLPGAR.F SEQ ID NO 1827 143-159 ADAMTS-4 G.LPGVYPGGVLPGAR.F SEQ ID NO 1829 146-159 ADAMTS-4 K.AGYPTGTGVGPQAAAAAAAK.A SEQ ID NO 1831 242-261 ADAMTS-4 G.GAGVPGVPGAIPGIGGIAGVG.T SEQ ID NO 1850 279-299 ADAMTS-4 G.AGVPGVPGAIPGIGGIAGVG.T SEQ ID NO 1851 280-299 ADAMTS-4 A.GVGTPAAAAAAAAAAK.A SEQ ID NO 1852 297-312 ADAMTS-4 G.VGTPAAAAAAAAAAK.A SEQ ID NO 1853 298-312 ADAMTS-4 G.GPGFGPGVVGVPGAGVPGVGVPG.A SEQ ID NO 1854 326-348 ADAMTS-4 G.ARPGVGVGGIPTYGVGA.G SEQ ID NO 1855 385-401 ADAMTS-4 A.RPGVGVGGIPTYGVGAG.G SEQ ID NO 1838 386-402 ADAMTS-4 A.RPGVGVGGIPTYGVGAGG.F SEQ ID NO 1856 386-403 ADAMTS-4 G.VGISPEAQAAAAAK.A SEQ ID NO 1841 435-448 ADAMTS-4 G.VGVAPGVGVAPGVGVAPGVGLAPGVG.V SEQ ID NO 1857 506-531 ADAMTS-4 A.PGVGVAPGVGLAPGVGVAPGVGVA.P SEQ ID NO 1858 516-539 ADAMTS-4 G.VGVAPGVGLAPGVGVAPGVG.V SEQ ID NO 1859 518-537 ADAMTS-4 L.APGVGVAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1860 527-551 ADAMTS-4 Y.GAAVPGVLGGLGALGGVGIPG.G SEQ ID NO 1861 658-678 ADAMTS-4 G.AAVPGVLGGLGALGGVGIPG.G SEQ ID NO 1848 659-678 ADAMTS-4 G.GAGQFPLGGVAARPGFGL.S SEQ ID NO 1862 750-767 ADAMTS-8 L.VPGGVADAAAAYK.A SEQ ID NO 1863 092-104 ADAMTS-8 G.VGLPGVYPGGVLPGAR.F SEQ ID NO 1828 144-159 ADAMTS-8 G.LPGVYPGGVLPGAR.F SEQ ID NO 1829 146-159 ADAMTS-8 P.GVYPGGVLPGAR.F SEQ ID NO 1830 148-159 ADAMTS-8 V.YPGGVLPGAR.F SEQ ID NO 1864 150-159 ADAMTS-8 F.GPGVVGVPGAGVPGVGVPG.A SEQ ID NO 1834 330-348 ADAMTS-8 G.ARPGVGVGGIPTYGVGA.G SEQ ID NO 1855 385-401 ADAMTS-8 V.APGVGVAPGVGVAPGVGLAPGVGV.A SEQ ID NO 1865 509-532 ADAMTS-8 L.APGVGVAPGVGVAPGVGV.A SEQ ID NO 1866 527-544 ADAMTS-8 L.APGVGVAPGVGVAPGVGVAPG.I SEQ ID NO 1867 527-547 ADAMTS-8 L.APGVGVAPGVGVAPGVGVAPGIG.P SEQ ID NO 1868 527-549 ADAMTS-8 L.APGVGVAPGVGVAPGVGVAPGIGP.G SEQ ID NO 1869 527-550 ADAMTS-8 L.APGVGVAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1860 527-551 ADAMTS-8 L.APGVGVAPGVGVAPGVGVAPGIGPGGVAA.A SEQ ID NO 1870 527-555 ADAMTS-8 G.VGVAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1871 530-551 ADAMTS-8 G.VAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1847 532-551 ADAMTS-8 G.AAVPGVLGGLGALGGVGIPG.G SEQ ID NO 1848 659-678 ADAMTS-8 G.AAVPGVLGGLGALGGVGIPGG.V SEQ ID NO 1872 659-679 ADAMTS-8 A.AVPGVLGGLGALGGVGIPG.G SEQ ID NO 1873 660-678 ADAMTS-8 A.VPGVLGGLGALGGVGIPGG.V SEQ ID NO 1874 661-679 ADAMTS-8 A.GQFPLGGVAARPGFGL.S SEQ ID NO 1875 752-767 Cat K G.ALVPGGVADAAAAYK.A SEQ ID NO 1876 090-104 Cat K G.LPYTTGKLPYGYGPG.G SEQ ID NO 1877 219-233 Cat K A.AAAAAAKAAAKFGA.G SEQ ID NO 1878 255-268 Cat K A.GVGTPAAAAAAAAAAK.A SEQ ID NO 1852 297-312 Cat K A.AAAAAAAAAKAAKYGA.A SEQ ID NO 1879 303-318 Cat K G.FGPGVVGVPGAGVPGVGVPG.A SEQ ID NO 1833 329-348 Cat K G.VGISPEAQAAAAAK.A SEQ ID NO 1841 435-448 Cat K G.VAPGVGVAPGVGVAPGVGLAPGVG.V SEQ ID NO 1880 508-531 Cat K G.VGVAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1871 530-551 Cat K G.VAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1847 532-551 Cat S T.FPGALVPGGVADAAAAYK.A SEQ ID NO 1881 087-104 Cat S G.VGLPGVYPGGVLPGAR.F SEQ ID NO 1828 144-159 Cat S G.LPGVYPGGVLPGARFPGVG.V SEQ ID NO 1882 146-164 Cat S G.YPTGTGVGPQAAAAAAAK.A SEQ ID NO 1883 244-261 Cat S G.GAGVPGVPGAIPGIGGIAGVG.T SEQ ID NO 1850 279-299 Cat S G.TPAAAAAAAAAAKAAK.Y SEQ ID NO 1884 300-315 Cat S G.VPGAGVPGVGVPGAGIPVVP.G SEQ ID NO 1885 336-355 Cat S G.VPGAGVPGVGVPGAGIPVVPGAGIPG.A SEQ ID NO 1886 336-361 Cat S G.ISPEAQAAAAAKAAK.Y SEQ ID NO 1887 437-451 Cat S V.PGVGVAPGVGVAPGVGVA.P SEQ ID NO 1888 504-521 Cat S G.VAPGVGVAPGVGVAPGIGPGGVA.A SEQ ID NO 1889 532-554 Cat S G.IPGGVVGAGPAAAAAAAK.A SEQ ID NO 1890 676-693 MMP1 G.GVLPGARFPGVGVLPGVPTGA.G SEQ ID NO 1891 153-173 MMP1 G.GVPGVGGVPGVGISPEA.Q SEQ ID NO 1892 425-441 MMP1 V.PGVGVAPGVGVAPGVGVA.P SEQ ID NO 1888 504-521 MMP1 G.VGVAPGVGVAPGVGVAPGVG.L SEQ ID NO 1893 506-525 MMP1 G.VAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1847 532-551 MMP1 A.AVPGVLGGLGALGGVGIPG.G SEQ ID NO 1873 660-678 MMP1 MMP3 G.ALVPGGVADAAAAYK.A SEQ ID NO 1876 090-104 MMP3 G.YPTGTGVGPQAAAAAAAK.A SEQ ID NO 1883 244-261 MMP3 G.VPGVPGAIPGIGGIAGVG.T SEQ ID NO 1894 282-299 MMP3 F.GPGVVGVPGAGVPGVGVPGA.G SEQ ID NO 1895 330-349 MMP3 G.VGISPEAQAAAAAK.A SEQ ID NO 1841 435-448 MMP3 G.VGVAPGVGVAPGVGLAPGVG.V SEQ ID NO 1844 512-531 MMP3 G.VAPGVGVAPGVGVAPGIGPG.G SEQ ID NO 1847 532-551 MMP8 P.GVYPGGVLPGAR.F SEQ ID NO 1830 148-159 MMP8 K.AGYPTGTGVGPQAAAAAAAK.A SEQ ID NO 1831 242-261 MMP8 G.VPGVPGAIPGIGGIAGVG.T SEQ ID NO 1894 282-299 MMP8 F.GPGVVGVPGAGVPGVGVPG.A SEQ ID NO 1834 330-348 MMP8 G.VPGVGVPGAGIPVVPGA.G SEQ ID NO 1896 341-357 MMP8 G.ARPGVGVGGIPTYGVG.A SEQ ID NO 1836 385-400 MMP8 A.RPGVGVGGIPTYGVGAG.G SEQ ID NO 1838 386-402 MMP8 G.VGVAPGVGVAPGVGVAP.G SEQ ID NO 1897 506-522 MMP8 G.VGVAPGVGVAPGVGLAPGVG.V SEQ ID NO 1844 512-531 MMP8 G.VGVAPGVGVAPGVGVAP.G SEQ ID NO 1897 530-546 MMP8 G.IPGGVVGAGPAAAAAAAK.A SEQ ID NO 1890 676-693 *Aminoacid residue numbers in the human elastin sequence

Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of elastin by a protease at an N- or C-terminal site, or where indicated a site marked by the sign in any one of the partial sequences of elastin in Table 24.

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of elastin.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

TABLE 25 N-terminal sequences of  protease generated peptide  fragments of elastin. Elastin GVPGAI SEQ ID NO 1898 ALGGGA SEQ ID NO 1901 PLKPVP SEQ ID NO 1904 GLGAGL SEQ ID NO 1907 LVPGGV SEQ ID NO 1910 PVGYPG SEQ ID NO 1913 VGPFGG SEQ ID NO 1916 TTGKLP SEQ ID NO 1919 FGAGAA SEQ ID NO 1922 AGIPGL SEQ ID NO 1925 TPAAAA SEQ ID NO 1928 VGVPGA SEQ ID NO 1931 GIPVVP SEQ ID NO 1934 ARPGVG SEQ ID NO 1937 SVGGVP SEQ ID NO 1940 VGVAPG SEQ ID NO 1943 GVPVAP SEQ ID NO 1946 LGAGIP SEQ ID NO 1949 PGALAA SEQ ID NO 1952 ALGGVG SEQ ID NO 1955 AGPAAA SEQ ID NO 1958 GLGVPG SEQ ID NO 1961 PLGGVA SEQ ID NO 1964 GVGLPG SEQ ID NO 1967 VPGVPV SEQ ID NO 1970 APGVGV SEQ ID NO 1973 GPGFGP SEQ ID NO 1976 VPGGVF SEQ ID NO 1979 LGPGGK SEQ ID NO 1982 LAGAGL SEQ ID NO 1985 LGAFPA SEQ ID NO 1988 LGVSAG SEQ ID NO 1991 FPGVGV SEQ ID NO 1994 PGVPLG SEQ ID NO 1996 YGPGGV SEQ ID NO 1999 GAGVPG SEQ ID NO 2002 IPGIGG SEQ ID NO 2005 VPGVGV SEQ ID NO 2008 AVPGVV SEQ ID NO 2011 GVGAGG SEQ ID NO 2014 FGLVPG SEQ ID NO 2017 PGVGLA SEQ ID NO 2020 LRAAAG SEQ ID NO 2023 GIPGLG SEQ ID NO 2026 AAAGLG SEQ ID NO 2029 VGIPGG SEQ ID NO 2032 GLVGAA SEQ ID NO 2035 GGVLGG SEQ ID NO 2038 GVAARP SEQ ID NO 2041 GVYPGG SEQ ID NO 2044 AAVPGV SEQ ID NO 2047 VPGVLG SEQ ID NO 2050 ISPEAQ SEQ ID NO 2053 LGALGG SEQ ID NO 2056 GKPLKP SEQ ID NO 2059 AGLGAG SEQ ID NO 2062 VTFPGA SEQ ID NO 2065 GLPGVY SEQ ID NO 2068 GAFAGI SEQ ID NO 2071 YTTGKL SEQ ID NO 2074 PQAAAA SEQ ID NO 2077 AIPGGV SEQ ID NO 1899 LGGGAL SEQ ID NO 1902 LKPVPG SEQ ID NO 1905 LGAGLG SEQ ID NO 1908 VADAAA SEQ ID NO 1911 ARFPGV SEQ ID NO 1914 GPQPGV SEQ ID NO 1917 LPYGYG SEQ ID NO 1920 GVLPGV SEQ ID NO 1923 VPGAIP SEQ ID NO 1926 PAAAAA SEQ ID NO 1929 AVGPGV SEQ ID NO 1932 IPGAAV SEQ ID NO 1935 RPGVGV SEQ ID NO 1938 VGGVPG SEQ ID NO 1941 VAPGVG SEQ ID NO 1944 GAGIPG SEQ ID NO 1947 PGFGPG SEQ ID NO 1950 AKYGAA SEQ ID NO 1953 LGGVGI SEQ ID NO 1956 GPAAAA SEQ ID NO 1959 LGGIPP SEQ ID NO 1962 LGGVAA SEQ ID NO 1965 VGLPGV SEQ ID NO 1968 VPGVGI SEQ ID NO 1971 VPGGVA SEQ ID NO 1974 YPTGTG SEQ ID NO 1977 GVFYPG SEQ ID NO 1980 GPGGKP SEQ ID NO 1983 AGAGLG SEQ ID NO 1986 AFPAVT SEQ ID NO 1989 VPGVGL SEQ ID NO 1992 KPGAPG SEQ ID NO 1995 GYPIKA SEQ ID NO 1997 AGYPTG SEQ ID NO 2000 AGVPGV SEQ ID NO 2003 IGGIAG SEQ ID NO 2006 VPVGVA SEQ ID NO 2009 VPGVVS SEQ ID NO 2012 VGAGGF SEQ ID NO 2015 GLVPGV SEQ ID NO 2018 GVGLAP SEQ ID NO 2021 LVGAAG SEQ ID NO 2024 LGVGVG SEQ ID NO 2027 AVPGVL SEQ ID NO 2030 IPGGVV SEQ ID NO 2033 VGAAGL SEQ ID NO 2036 GAGQFP SEQ ID NO 2039 VAARPG SEQ ID NO 2042 LPYTTG SEQ ID NO 2045 AAGLGG SEQ ID NO 2048 GQFPLG SEQ ID NO 2051 GVLPGA SEQ ID NO 2054 VPGVPG SEQ ID NO 2057 IAGVGT SEQ ID NO 2060 VVGVPG SEQ ID NO 2063 AGIPVV SEQ ID NO 2066 GARPGV SEQ ID NO 2069 VAGVPS SEQ ID NO 2072 PGVGVA SEQ ID NO 2075 LAPGVG SEQ ID NO 2078 GVPGGV SEQ ID NO 1900 GGALGP SEQ ID NO 1903 GLAGAG SEQ ID NO 1906 AGLGAF SEQ ID NO 1909 KAAKAG SEQ ID NO 1912 RFPGVG SEQ ID NO 1915 PQPGVP SEQ ID NO 1918 GYGPGG SEQ ID NO 1921 VLPGVG SEQ ID NO 1924 AIPGIG SEQ ID NO 1927 AAAAAA SEQ ID NO 1930 GVPGVG SEQ ID NO 1933 GAAVPG SEQ ID NO 1936 VGGIPT SEQ ID NO 1939 GVGTPA SEQ ID NO 1942 GVAPGV SEQ ID NO 1945 PGGVAA SEQ ID NO 1948 PGVVGV SEQ ID NO 1951 YGAAVP SEQ ID NO 1954 GVGIPG SEQ ID NO 1957 FGLVGA SEQ ID NO 1960 LGGVLG SEQ ID NO 1963 GGVAAR SEQ ID NO 1966 LPGVYP SEQ ID NO 1969 VGISPE SEQ ID NO 1972 YPGGVL SEQ ID NO 1975 VPGAGV SEQ ID NO 1978 VFYPGA SEQ ID NO 1981 PGGKPL SEQ ID NO 1984 GAGLGA SEQ ID NO 1987 AVTFPG SEQ ID NO 1990 PGVGLP SEQ ID NO 1993 PKAPGV SEQ ID NO 1493 PKLPGG SEQ ID NO 1998 TGVGPQ SEQ ID NO 2001 GVPGVP SEQ ID NO 2004 GIAGVG SEQ ID NO 2007 VPGAGI SEQ ID NO 2O1O YGARPG SEQ ID NO 2013 VGVGGI SEQ ID NO 2016 LVPGVG SEQ ID NO 2019 VGLAPG SEQ ID NO 2022 LVPGGP SEQ ID NO 2025 VGVPGL SEQ ID NO 2028 VLGGLG SEQ ID NO 2031 VVGAGP SEQ ID NO 2034 LGGLGV SEQ ID NO 2037 AFQFPL SEQ ID NO 2040 RPGFGL SEQ ID NO 2043 FGPGVV SEQ ID NO 2046 FPGALV SEQ ID NO 2049 ALVPGG SEQ ID NO 2052 VGAGVP SEQ ID NO 2055 GGLGAL SEQ ID NO 2058 VGAGPA SEQ ID NO 2061 LGVGGL SEQ ID NO 2064 FPLGGV SEQ ID NO 2067 PGFGLS SEQ ID NO 2070 GPGVVG SEQ ID NO 2073 VGTPAA SEQ ID NO 2076 or with any of the following sequences at the C-terminal of a peptide:

TABLE 26 C-terminal sequences of  protease generated peptide  fragments of Elastin. Elastin PGGVPG SEQ ID NO 2079 GALGGG SEQ ID NO 2081 GLGAFP SEQ ID NO 2083 VPGGVA SEQ ID NO 1974 RFPGVG SEQ ID NO 1915 PKAPGV SEQ ID NO 1493 LPYGYG SEQ ID NO 1920 GIAGVG SEQ ID NO 2007 AAAAKA SEQ ID NO 2091 GVGVPG SEQ ID NO 2092 VGGIPT SEQ ID NO 1939 GVGVGG SEQ ID NO 2097 GFPGFG SEQ ID NO 2099 PGVGIS SEQ ID NO 21O1 PGVGVA SEQ ID NO 2075 GIGPGG SEQ ID NO 2105 RAAAGL SEQ ID NO 2108 GVPGLG SEQ ID NO 2110 VLGGLG SEQ ID NO 2031 PAAAAA SEQ ID NO 1929 GLGVPG SEQ ID NO 1961 RPGFGL SEQ ID NO 2043 LSPIFP SEQ ID NO 2119 GPGGVA SEQ ID NO 2122 GLGALG SEQ ID NO 2123 GALGPG SEQ ID NO 2125 AFPAVT SEQ ID NO 1989 AAAAYK SEQ ID NO 2128 PGVPTG SEQ ID NO 2131 PIKAPK SEQ ID NO 2134 VGTPAA SEQ ID NO 2076 GIGGIA SEQ ID NO 2137 IPVVPG SEQ ID NO 2139 GAGIPG SEQ ID NO 1947 PTYGVG SEQ ID NO 2142 IPTYGV SEQ ID NO 2145 AGGFPG SEQ ID NO 2147 VGVAPG SEQ ID NO 1943 PGVGLA SEQ ID NO 2020 GVAPGV SEQ ID NO 1945 GLGVGG SEQ ID NO 2153 GLGVGA SEQ ID NO 2155 VGAGPA SEQ ID NO 2061 GGLGVG SEQ ID NO 2158 AGQFPL SEQ ID NO 2160 GFGLSP SEQ ID NO 2161 AAKFGA SEQ ID NO 2164 AGLGAL SEQ ID NO 2167 LKPVPG SEQ ID NO 1905 ALVPGG SEQ ID NO 2052 VLPGAR SEQ ID NO 2172 PTGAGV SEQ ID NO 2175 AGAAGK SEQ ID NO 2176 PGAGLG SEQ ID NO 2080 LGGGAL SEQ ID NO 1902 LGAFPA SEQ ID NO 1988 ADAAAA SEQ ID NO 2084 VGVLPG SEQ ID NO 2086 GVGPFG SEQ ID NO 2088 AAAAAK SEQ ID NO 2090 AIPGIG SEQ ID NO 1927 VVGVPG SEQ ID NO 2063 PVVPGA SEQ ID NO 2093 GGIPTY SEQ ID NO 2095 GVGGIP SEQ ID NO 2098 FGVGVG SEQ ID NO 2100 SPEAQA SEQ ID NO 2102 GVGVAP SEQ ID NO 2103 APGIGP SEQ ID NO 2106 AAAGLG SEQ ID NO 2029 GVPGFG SEQ ID NO 2111 VGIPGG SEQ ID NO 2032 VPGVGG SEQ ID NO 2114 PGVGGL SEQ ID NO 2116 GVAARP SEQ ID NO 2041 AQAAAA SEQ ID NO 2120 TPAAAA SEQ ID NO 1928 LGALGG SEQ ID NO 2056 VAPVGV SEQ ID NO 2126 AVTFPG SEQ ID NO 1990 AAKAGA SEQ ID NO 2129 GVPTGA SEQ ID NO 2132 KLPGGY SEQ ID NO 2135 VPGVPG SEQ ID NO 2057 GTPAAA SEQ ID NO 2138 VGVPGA SEQ ID NO 1931 PEAAAK SEQ ID NO 2141 TYGVGA SEQ ID NO 2143 PGAIPG SEQ ID NO 2146 GIPGVA SEQ ID NO 2148 VGLAPG SEQ ID NO 2022 LAPGVG SEQ ID NO 2078 GGVAAA SEQ ID NO 2151 PGLGVG SEQ ID NO 2154 ALAAAK SEQ ID NO 2156 AGPAAA SEQ ID NO 1958 LGVGGL SEQ ID NO 2064 GGVAAR SEQ ID NO 1966 PIFPGG SEQ ID NO 2162 AAKYGA SEQ ID NO 2165 GAGVPG SEQ ID NO 2002 PGVGVG SEQ ID NO 2169 RPGVGV SEQ ID NO 1938 GGFPGF SEQ ID NO 2173 VGGVPG SEQ ID NO 1941 GAGLGA SEQ ID NO 1987 GGGALG SEQ ID NO 2082 LGAGLG SEQ ID NO 1908 PGVLGG SEQ ID NO 2085 VPTGAG SEQ ID NO 2087 VPLGYP SEQ ID NO 2089 IPGIGG SEQ ID NO 2005 IGGIAG SEQ ID NO 2006 GVPGVG SEQ ID NO 1933 VVSPEA SEQ ID NO 2094 GIPTYG SEQ ID NO 2096 VGVPGL SEQ ID NO 2028 GVGAGG SEQ ID NO 2014 VAPGVG SEQ ID NO 1944 APGVGL SEQ ID NO 2104 VAPGIG SEQ ID NO 2107 AAGLGA SEQ ID NO 2109 AGVPGL SEQ ID NO 2112 GAGPAA SEQ ID NO 2113 GLGGLG SEQ ID NO 2115 PAAAAK SEQ ID NO 2117 AARPGF SEQ ID NO 2118 GPGIPG SEQ ID NO 2121 PGGVAA SEQ ID NO 1948 ALGPGG SEQ ID NO 2124 KPVPGG SEQ ID NO 2127 VTFPGA SEQ ID NO 2065 VPQPGA SEQ ID NO 2130 AGVKPK SEQ ID NO 2133 YGYGPG SEQ ID NO 2136 GVGTPA SEQ ID NO 1942 AAAAAA SEQ ID NO 1930 GVPGAG SEQ ID NO 2140 ARPGVG SEQ ID NO 1937 YGVGAG SEQ ID NO 2144 VGAGGF SEQ ID NO 2015 GISPEA SEQ ID NO 2149 VPGAPG SEQ ID NO 2150 APGVGV SEQ ID NO 1973 PGIGPG SEQ ID NO 2152 LGVGVG SEQ ID NO 2027 LGGLGA SEQ ID NO 2157 GPAAAA SEQ ID NO 1959 GVGGLG SEQ ID NO 2159 VAARPG SEQ ID NO 2042 IFPGGA SEQ ID NO 2163 AAKAAK SEQ ID NO 2166 GIPGGV SEQ ID NO 2168 IPPAAA SEQ ID NO 2170 ARPGFG SEQ ID NO 2171 GLSPIF SEQ ID NO 2174 GIPVVP SEQ ID NO 1934

Vimentin

Several candidate proteases may be responsible for the digestion of vimentin in fibrotic tissue We have through a range of in vitro cleavages of pure native proteins determined that the enzymes listed in the following table cleaved vimentin at least at the cleavage sites at each end of the following sequences or at the cleavage sites marked ‘.’ or where no ‘.’ is shown, at the ends of the sequences:

TABLE 27 Vimentin fragments generated by specific proteases. Aminoacid residue Protease Sequence between cleavage sites numbers* MMP2, RLRSSVPGVR. SEQ ID NO 2177 69-78 MMP8, Trypsin MMP2, RLRSSVPGVL. SEQ ID NO 2178 69-78 MMP8, Trypsin MMP2, .LLQDSVDFSL SEQ ID NO 2179 79-89 MMP8, Trypsin MMP2, .FADLSEAANR SEQ ID NO 2180 295-304 MMP8, Trypsin MMP2 .ISLPLPTFSS SEQ ID NO 2181 410-420 *in the human vimentin sequence

Accordingly, in a method of the invention, said peptide fragments preferably comprise a neo-epitope formed by cleavage of vimentin by a protease at an N- or C-terminal site, or where indicated a site marked by the sign in any one of the partial sequences of vimentin in Table 24.

The immunological binding partner may be one specifically reactive with a C-terminal or N-terminal neo-epitope formed by cleavage of vimentin.

Suitable immunological binding partners may therefore be specifically reactive with any of the following sequences at the N terminal of a peptide:

TABLE 28 N-terminal sequences   of protease generated peptide fragments  of vimentin. Vimentin LLQDSV SEQ ID NO 2182 FADLSE SEQ ID NO 2183 ISLPLP SEQ ID NO 2184 or with any of the following sequences at the C-terminal of a peptide:

TABLE 29 C-terminal sequences  of protease generated peptide fragments  of vimentin. Vimentin SVPGVR SEQ ID NO 2185 SVPGVL SEQ ID NO 2186

Further cleavage sites defining neo-epitopes that may be assayed in a similar manner can be identified by exposing collagens, elastin, CRP and proteoglycans or other fibrotic tissue proteins to any of the enzymes described herein and isolating and sequencing peptides thereby produced. Furthermore, assays may be based on the neo-epitopes generated adjacent the illustrated cleavage sites, i.e. in the C-terminal sequences that lead up to the N-terminal epitopes given above and the N-terminal sequences that connect to the C-terminal epitopes described.

Assays for more than one of the peptides described above may be conducted separately and their results combined or more than one of the peptides described above may be measured together.

The result of an assay according to the invention may be combined with one or more other measured biomarkers to form a composite index of diagnostic or prognostic value.

Generally, all previously known immunoassay formats can be used in accordance with this invention including heterogeneous and homogeneous formats, sandwich assays, competition assays, enzyme linked assays, radio-immune assays and the like. Thus, optionally, said method is conducted as a competition immunoassay in which said immunological binding partner and a competition agent are incubated in the presence of said sample and the competition agent competes with the peptide fragments in the sample to bind to the immunological binding partner.

Said competition agent may be (1) a synthetic peptide derived from the sequence of collagen type I, III, IV, V, or VI, or from CRP, or from any of the proteoglycans versican, lumican, perlecan, decorin and biglycan peptide, or a competition agent derived from (2) a purified native collagen type I, III, IV, V, or VI, or CRP, or any of the proteoglycans neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, perlecan, decorin and biglycan cleaved by proteases to reveal said neo-epitope.

One suitable method could be a competition immunoassay using monoclonal antibodies or antibody binding fragments binding to neo-epitopes of collagen type I, III, IV, V, VI, CRP, vimentin, or any of the proteoglycans neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan fragments or neo-epitopes on peptide fragments from other proteins derived from fibrotic tissue. Appropriately selected synthetic peptides coated onto the solid surface of a microtitre plate could compete with the sample for binding to the monoclonal antibodies or binding fragments. Alternatively, purified, native collagen type I, III, IV, V, VI, CRP, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan fragments carrying the neo-epitope recognised by the monoclonal antibody or binding fragment could be used on the solid surface. Yet another alternative is to immobilise the monoclonal antibody or binding fragment on the solid surface and then co-incubate the sample with a synthetic peptide appropriately linked to a signal molecule, e.g. horseradish peroxidase or biotin.

The sample may be a sample of serum, blood, plasma or other, e.g. fibrotic tissue biopsy.

Assays may be conducted as sandwich assays using a first immunological binding partner specifically reactive with a said neo-epitope and a second immunological binding partner reactive with the relevant protein to which the neo-epitope belongs. Optionally, said second immunological binding partner is directed to a second neo-epitope of the same protein.

In certain preferred methods the method further comprises comparing the determined level of said binding of said peptide fragments with values characteristic of (a) comparable healthy individuals and/or (b) a pathological fibrotic condition and optionally associating a higher level of the measured peptide (normally indicated by a higher level of binding) with a more severe degree of a said condition.

An aspect of the present invention relates to the development of monoclonal antibodies recognising neo-epitopes as described above, especially for collagen types I and IV. This can be achieved by immunising mice with synthetic peptides originating from the amino acid sequence of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan molecules (including the sequences listed above or sequences terminating therein), fusing the spleen-cells from selected mice to myeloma cells, and testing the monoclonal antibodies for binding to neo-epitopes on relevant synthetic peptides. Specificity for neo-epitopes can be ensured by requiring reactivity with a synthetic peptide and a lack of reactivity with either a C-prolongated form of the immunising peptide (for a C-terminal neo-epitope) or an N-terminal prolongated form of the immunising peptide (for an N-terminal neo-epitope). Antibodies for neo-epitopes may also be evaluated to establish a lack of binding capacity to native collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, pelecan and biglycan. Alternatively, specificity for a neo-epitope can be ensured by requiring the reactivity of the antibody to be negatively dependent on the presence of biotin or other functional groups covalently linked to one of the terminal amino acids.

The invention includes an immunological binding partner which is specifically immunoreactive with a neo-epitope formed by cleavage of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan by a protease at a end-site in any one of the partial sequences of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan set out above, and may be for instance a monoclonal antibody or a binding fragment thereof.

The invention includes a cell line producing a monoclonal antibody against a C-terminal or N-terminal neo-epitope formed by cleavage of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan at the end-sites of sequences in any one of the partial sequences of collagen type I, III, IV, V, VI, CRP, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan set out above.

The invention further provides a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan in any one of the partial sequences of these proteins set out above. Such a peptide may be conjugated as a hapten to a carrier for producing an immune response to said peptide, or immobilised to a solid surface or conjugated to a detectable marker for use in an immunoassay.

The invention further comprises an isolated nucleic acid molecule coding for a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan in any one of the partial sequences of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan set out above.

The invention further comprises a vector comprising a nucleic acid sequence comprising an expression signal and a coding sequence which codes for the expression of a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of collagen type I, III, IV, V, VI, CRP, vimentin, versican, lumican, decorin, perlecan and biglycan in any one of the partial sequences of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan set out above and further includes a host cell transformed with such a vector and expressing a said peptide.

Yet another aspect of the invention relates to kits, which can be used conveniently for carrying out the methods described above. Such kits may include (1) a microtitre plate coated with synthetic peptide; (2) a monoclonal antibody or antibody binding fragment of the invention reactive with said synthetic peptide; and (3) a labelled anti-mouse IgG immunoglobulin. Alternatively, such kits may include (1) a microtitre plate coated with purified native collagen type I, III, IV, V, VI, CRP, vimentin, versican, lumican, decorin, perlecan and biglycan fragments; (2) a monoclonal antibody recognising a neo-epitope on collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan fragments and reactive with said purified collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan, and biglycan fragments; and (3) a labelled anti-mouse IgG immunoglobulin. Alternatively, such kits may include (1) a microtitre plate coated with streptavidin; (2) a synthetic peptide linked to biotin; (3) a monoclonal antibody recognising a neo-epitope on collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan fragments and reactive with said synthetic peptide; and (4) a labelled anti-mouse IgG immunoglobulin. Yet another alternative could be kits including (1) a microtitre plate coated with streptavidin; (2) a synthetic peptide linked to biotin; (3) a monoclonal antibody recognising a neo-epitope on collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan fragments (and reactive with said synthetic peptide) and conjugated to horseradish peroxidase.

Thus, the invention includes an immunoassay kit comprising an immunological binding partner as described herein, especially in respect of collagens types I and IV, and a competition agent which binds said immunological binding partner, and optionally one or more of a wash reagent, a buffer, a stopping reagent, an enzyme label, an enzyme label substrate, calibration standards, an anti-mouse antibody and instructions.

The assays described herein are useful in the diagnosis of fibrosis in patients. In addition, the tests are useful for the assessment of disease progression, and the monitoring of response to therapy. The immunological binding partners of the invention may also be used in immunostaining to show the presence or location of collagen type I, III, IV, V, VI, CRP, vimentin, neurocan, brevican, fibromodulin, serglycins, syndecan, betaglycan, versican, lumican, decorin, perlecan and biglycan cleavage products.

EXEMPLIFICATION Example 1 Collagen Type III Degraded with MMP-9

Method

Cleavage: Collagen type III isolated from human placenta was dissolved in 10 mM acetic acid (1 mg/ml). The protein solution was then passed through a filter (Microcon Ultracel YM-10) to remove fragment contaminations. MMP-9 was preactivated with 4-aminophenylmercuric acetate (APMA, Sigma) at 37° C. for 3 hours. After activations, collagen type III and MMP-9 were mixed 100:1 and incubated shaking for 3 days at 37° C.

The solution was analyzed by liquid chromatography/mass spectrometry (LC/MS) and the fragments were identified by performing Mascot Search. The peptide sequences were selected by homology search, ensuring no cross-reactivity to other or related proteins, as well as interspecies cross-reactivity.

Antibody design: The peptide sequences were synthesized and conjugated to ovalbumin (OVA). Mice were immunized ever 2-3 weeks, up to five. Antibody titers were checked by screening peptides, both selection and de-selection. When sufficient antibody titers were achieved, positive mice were selected for fusion, euthanized, and the spleen was disintegrated and B-cells were removed for fusion with myeloma cells. Selections of antibody producing cells were done by culturing and re-seeding the surviving chimera cells in single cell clones. Clones are selected by selection and de-selection peptides followed by native reactivity testing (FIG. 1), as neoepitopes are generated by synthetic small peptide sequences, which may not reflect the native proteins. An IgG subtype clone is selected for antibody production. Antibody purification is done by protein-G column.

Assay development: Optimal antibody concentrations are determined by checker-board analysis, with dilutions of antibody coating and screening peptide, in competitions ELISA. The different determination for the collagen degraded by MMP-9 (CO3) assay is shown in Table 30.

TABLE 30 Limit of Detection, Avarage Inter- and Intraassay variation of the CO3 assay. Limit of Detection 0.5 ng/ml Average Interassay variation 3.71% Average Intraassay variation 5.48%

Example 2 CO3 in Biological Relevant Samples

CO3 Levels in Bile Duct Ligated Rats Compared to Sham Operated Rats.

Method: Forty female Sprague-Dawley rats (6 months old) were housed at the animal research facilities at Nordic Bioscience. The experiments were approved by the Experimental Animal Committee of the Danish Ministry of Justice, and were performed according to the European Standard for Good Clinical Practice (2008/561-1450). The rats were housed in standard type III-H cages at 18-22° C. with bedding and nest material (Altromin 1324; Altromin, Lage, Germany) and purified water (Milli-Q system; Millipore, Glostrup, Denmark) ad libitum. Rats were kept under conditions of a 12-hour light/dark cycle.

Liver fibrosis was induced by common BDL. In short: The rat was anaesthetized, the bile duct found, two ligations were performed around the bile duct followed by dissection between the ligations, the abdomen was closed. In sham operated rats, the abdomen was closed without bile duct ligation.

The rats were divided into 2 groups: Group 1(10 BDL and 10 sham operated rats) were sacrificed after 2 weeks, and Group 2 (9 BDL and 10 sham operated rats) were sacrificed after 4 weeks. On completion of the study period (2, or 4 weeks), after at least 14 hours fasting, all surviving animals were asphyxiated by CO₂ and sacrificed by exsanguinations.

Blood samples were taken from the retro-orbital sinus of at least 14 hours fasting rats under light CO₂/O₂ anaesthesia at baseline and at termination. The blood were collected and left 30 minutes at room temperature to cloth, followed by centrifugation at 1500 g for 10 minutes. All clot-free liquid were transferred to new tubes and centrifuged again at 1500 g for 10 minutes. The serum were then transferred to clean tubes and stored at −80° C.

CO3 were measured in ×5 diluted serum samples from the rats. Sham and BDL levels were compared by Mann-Whitneys two-tailed nonparametric test (α=0.05) of statistical significance assuming normal distribution.

CO3 levels increased significantly in the BDL groups compared to the Sham-operated animals. The results are shown in FIGS. 2 a and b.

Example 3 CO3 in Different Fibrotic Diseases (Human Serum)

CO3 levels were measured in serum from human with three different fibrotic diseases: Chronic obstructed pulmonary disease (COPD), Scleroderma, and Hepatitis virus C (HCV). The serum samples were retrieved from Sera Laboratories International Ltd (SLI Ltd), UK.

CO3 levels were increased in the three different fibrotic diseases (FIG. 3)

Example 4 Antibody Development—Detection of Marker CO3-610C

Type III collagen (Abcam, Cambridge, UK) was degraded in vitro by activated MMP-9 (Merck KGaA, Darmstadt, Germany) for 2 days. Degradation fragments were sequenced by LS-MS/MS and identified by MASCOT search. A specific peptide sequence ⁶¹⁰KNGETGPQ (SEQ ID NO2251) was selected for antibody production. The N-terminal of this sequence is residue 610 of human collagen type III. The synthetic peptide was conjugated to ovalbumin prior to subcutaneous immunization of 4-6 week old Balb/C mice with about 200 μL emulsified antigen and 50 μg CO3-610C (KNGETGPQGPGGC(SEQ ID NO2252)-OVA). Consecutive immunizations were performed at two week intervals until stable sera titer levels were reached in Freund's incomplete adjuvant. The mice were bled from the second immunization on. At each bleeding, the serum titer was measured and the mouse with highest anti-serum titer was selected for fusion. After the fourth immunization, this mouse was rested for one month and then boosted intravenously with 50 μg CO3-610C in 100 μL 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion.

Monoclonal antibody producing clones were selected using a) immunogenic peptide: KNGETGPQGP-GGC(SEQ ID NO2253)-Ovalbumine (OVA) (807678), b) screening peptide KNGETGPQGP-PG-K(SEQ ID NO2254)-Biotin (807971), c) de-selection peptides KDGETGAAGPPGK(SEQ ID NO2255)-Biotin (118318) representing a type II collagen alpha 1 chain, KDGEAGAQGPPGK(SEQ ID NO2256)-Biotin representing a type I collagen alpha 1 chain degradation product, purchased from the Chinese Peptide Company, Beijing, China. The ELISA coat plate was obtained from NUNC (Thermofisher, Copenhagen, Denmark). Peptide conjugation reagents and buffers were produced by Pierce (Thermofisher, Copenhagen, Denmark).

Buffer used for dissolving the coating peptide was composed of the following: 40 mM Na₂HPO₄, 12 H₂O, 7 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, 25 mM EDTA, 0.1% Tween 20, 1% BSA, 10% sorbitol, pH 7. For a serum assay, buffer containing the following chemicals was used: 8 mM Na₂HPO₄, 12 H₂O, 1.5 mM KH₂PO₄, 13.7 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 1% BSA, 0.003% phenol red, pH 7.4. A different buffer used for a urine assay contained 400 mM TRIZMA, 0.05% Tween 20, 0.1% BSA, 0.36% Bronidox L5, pH 8.0. For both serum and urine assays we used a washing buffer composed of 25 mM TRIZMA, 50 mM NaCl, 0.036% Bronidox L5, 0.1% Tween 20, and reaction-stopping buffer composed of 0.1% H₂SO₄. ELISA-plates used for the assay development were Streptavidin-coated from Roche (Hvidovre, Denmark) cat.: 11940279. All ELISA plates were analyzed with the ELISA reader from Molecular Devices, SpectraMax M, (CA. USA).

In preliminary experiments, we optimized the reagents, their concentrations and the incubation periods by performing several checkerboard analyses. A 96-well ELISA plate coated with streptavidin was further coated with 5 ng/ml of the synthetic peptide KNGETGPQGP(SEQ ID NO2257)-Biotinylated dissolved in PBS-TBE buffer at 20° C. for 30 minutes by constant shaking at 300 rpm. After washing with washing buffer, 20 μl of sample was added, followed by 100 μl of peroxidase conjugated anti-human mAb-NB51-32 CO3-610C solution (23 pg/ml in incubation buffer). The plate was incubated for 1 hour at 20° C. during which time it was shaken at 300 rpm. This was followed by washing and finally, 100 μl tetramethylbenzinidine (TMB) (Kem-En-Tec cat.4380H) was dispensed and the plate incubated for 15 minutes in darkness and shaken at 300 rpm. In order to cease the reaction, 100 μl of stopping solution was added and the plate analyzed in the ELISA reader at 450 nm with 650 nm as reference.

A standard curve was performed by serial dilution of biotinylated-NB51-32 CO3-610C for a serum assay, and biotinylated-NB51-134 CO3-610C for a urine assay. Standard concentrations were 0, 0.33, 1, 3, 9, 27, 81 and 162 ng/ml.

We designate fragments detected using the immunoassays so obtained as CO3-610C as the amino acid K at the N-terminal of the sequence KNGETGPQGP(SEQ ID NO2257) is amino acid 610 of the human collagen III sequence.

Example 5 Comparison of CO3-610C and Other Biomarkers in Induced Liver Fibrosis in Rats

Animals

40 female Sprague-Dawley rats aged 6 months were housed at the animal research facilities at Nordic Bioscience, Copenhagen, Denmark. The experiments were approved by the Experimental Animal Committee of the Danish Ministry of Justice and were performed according to the European Standard for Good Clinical Practice (2008/561-1450). The rats were housed in standard type III-H cages at 18-22° C. with bedding and nest material (Altromin 1324; Altromin, Lage, Germany) and water ad libitum. Rats were kept under conditions of a 12-hour light/dark cycle.

Study Design

In 20 rats, liver fibrosis was induced by common BDL. The surgical procedure was performed under sterile conditions. The rat was anaesthetized, the bile duct localized and ligated in two places followed by dissection between the ligations, and the abdomen was closed. The other 20 rats were subjected to a sham operation, in which the abdomen was closed without bile duct ligation. The rats were then divided into 2 groups: Group 1 (10 BDL rats and 10 sham-operated rats) was sacrificed after 2 weeks and Group 2 (10 BDL and 10 sham-operated rats) was sacrificed after 4 weeks. On completion of the study period (2 or 4 weeks), after at least 14 hours fasting, all surviving animals were asphyxiated by CO₂ and sacrificed by exsanguinations.

Blood Sampling

Blood samples were taken from the retro-orbital sinus of rats after at least 14 hours fasting, under light CO₂/O₂ anaesthesia, at baseline and at termination. Blood was left 30 minutes at room temperature to clot, followed by centrifugation at 1500 g for 10 minutes. All clot-free liquid was transferred to fresh tubes and centrifuged again at 1500 g for 10 minutes. The serum was then transferred to clean tubes and stored at −80° C.

Tissue Handling

After the rats were put down, their livers were carefully dissected, weighed, fixed in 4% formaldehyde for a minimum of 24 hours, cut into appropriate slices and embedded in paraffin. Sections 5 μthick were cut, mounted on glass slides and stained with Sirius Red. The liver sections were evaluated histologically by assessment of the architecture, presence of inflammation, proliferation of bile ducts and fibrosis. The de novo bile duct formation in the parenchyma was evaluated semi-quantitatively using the following scoring system: normal=0, mild changes (⅓ or less of the lobule affected)=1, moderate changes (between ⅓ and ⅔ of the lobule affected)=2, and severe changes (⅔ or more of the lobule affected)=3. Digital photographs were captured using an Olympus BX60 microscope with ×40 and ×100 magnification and an Olympus 5050-zoom digital camera (Olympus, Tokyo, Japan).

Determination of Total Collagen and Serum CTX-II

The total collagen concentration was assayed using the commercial QuickZyme Collagen Assay (QuickZyme Bioscience, Leiden, The Netherlands). The concentration of CTX-II was assayed using the commercial Rat CTX-II kit (IDS Nordic, Herlev, Denmark). All samples were assayed in duplicate.

Type III Collagen mRNA Quantification

The number of transcripts of type III collagen (Col3a1) in liver tissue samples was determined by quantitative real-time polymerase chain reaction (RT-PCR) using fluorescent reporter probes. The number of Col3a1 copies in the sample was extrapolated from a standard curve obtained using Col3a1 plasmid cDNA Image Clone 7097081 (Geneservice, Cambridge, UK) as dilution standard. Amounts of Col3a1 were normalized with those of housekeeping gene hypoxanthine phosphoribosyltransferase 1 (Hprt1). Primers and probes for Col3a1 and Hprt1 mRNAs were designed using NCBI Reference Sequences NM_(—)032085.1 and NM_(—)012583.2 as templates, respectively (TIB Molbiol GmbH, Berlin, Germany). Total RNA was extracted from frozen liver samples using Absolutely RNA Miniprep kit (Stratagene, La Jolla, Calif., USA) following the manufacturer's instructions and its quality assessed in RNA Nano chips using a 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, Calif., USA). Immediately after RNA isolation, complementary DNA (cDNA) was synthesised with Transcriptor First Strand cDNA Synthesis kit (Roche, Basel, Switzerland) using 1 μg of RNA as the template. For each sample tested, a cDNA synthesis negative control, omitting reverse transcriptase enzyme from the reaction mix, was included. Separate PCR reactions for Col3a1 and Hprt1 were performed in a 20 μL format using the Lightcycler Faststart DNA Master Plus Hybprobe kit (Roche) according to the manufacturer's instructions. Real time fluorescence data was collected in a Lightcycler 2.0 instrument (Roche).

Extractions

Tissue was pulverized in excess of liquid nitrogen in a steel mortar. Samples were then transferred into a 1.5 ml eppendorf tube and left shaking overnight at 4° C. in 0.5M Acetic Acid solution containing protease inhibitor cocktail (Roche). The samples were then sonicated with ultrasounds using 5 pulses at 60% amplitude (U50 control, IKA Labortechnik) and left for an additional 2 hours at 4° C. after which they were centrifuged for 5 minutes at 13,000 rpm. Supernatant was carefully removed, transferred in a new eppendorf and stored at −80° C.

Densitometry

Densitometry measurements were performed using UN-SCAN-IT Version 6.1 from Silk Scientific (give city, country).

Histology Image Analysis

Histology sections stained with Sirius Red were analyzed using Visiopharm software Version 3.2.8.0 (give city, country). Images were acquired using Pixelink PL-A623C microscope digital camera.

SDS PAGE and Western Blots

20 μg of tissue extract was mixed with loading buffer (Invitrogen LDS 4×, NP0007) containing the reducing agent (NuPAGE, NP0004 from Invitrogen). Samples were then loaded into 4-12% Bis-Tris gradient gel (NP0332BOX from Invitrogen) and run for 52 minutes at 200V. Proteins were then transferred onto a nitrocellulose membrane using the i-Blot transfer system (Invitrogen), blocked with 5% milk in (? Need to spell out?) TTBS overnight at 4 degrees. Beta Actin antibody (AbCam ab8229, give company, city country?) was used as a loading control.

Statistical Analysis

Mean values and standard error of the mean (SEM) were calculated using GraphPad Prism (GraphPad Software, Inc., San Diego, Calif., USA) and compared by Student's two-tailed paired t-test (α=0.05) of statistical significance assuming normal distribution, or by Mann-Whitney two-tailed non-parametric test (α=0.05). The coefficient of correlation (R²) and the corresponding p-value was determined by linear regression.

Results

Liver Appearance:

At the time of sacrifice, livers of control animals showed normal gross morphology while livers of BDL animals were enlarged. The liver weights were significantly increased in BDL rats compared to the sham-operated controls (mean weights at sacrifice: 2 weeks post-surgery, sham 8.1 g; BDL 14.1 g; 4 weeks post-surgery, sham 9.0 g; BDL 19.4 g) (FIG. 4 panel A). Semi-quantitative scoring of liver sections using the 0-3 scale showed significantly more structural changes of the liver at 4 weeks compared with 2 weeks (FIG. 4 panel B). FIG. 4, panel A shows liver weight in bile duct ligation (BDL)- or sham-operated rats. Data are shown as mean+SEM. [***, P<0.0001. Panel B shows scoring of the structural changes in the liver of each group. Data are shown as mean+SEM. **, P=0.0094. Panel C shows Sirius Red photomicrographs showing the hepatic structure in sham-operated rats, and in BDL rats 2 and 4 weeks post-surgery. The hepatic structure around the portal tract is clearly disrupted in BDL rats compared with the sham-operated rats. Collagens are highlighted in red. Original magnification was ×40.

Under histological examination, the livers of sham-operated animals showed no sign of fibrosis and were microscopically normal (FIG. 4C). In the BDL livers, a marked ductal proliferation was observed. In the 2-week post-surgery group, the proliferation was located around the portal tract while in the 4-week group the proliferation had spread (FIG. 4C). Collagen deposition was found around the ductular structures. Inflammation was minimal and confined to the portal tracts. No signs of cholestasis were seen, whether intracellular cholestasis, bile plugs, bile infarctions or hepatocytic rosette formation.

Changes in CO3-610C Levels:

FIG. 5 shows in panel A MMP-9 mediated CO3 degradation serum levels in bile duct ligated (BDL)- or sham-operated rats. Data are shown as mean+standard error of mean. 2 weeks post-surgery *** P<0.0001 and 4 weeks post-surgery ** P=0.0014. In panel B are shown CO3-610C delta values (termination-baseline paired), 2 weeks post-surgery P<0.0001 and 4 weeks post-surgery P=0.0016. In panel C are shown CTX-II levels in BDL- or sham-operated rats. Data are shown as mean+standard error of mean.

In the BDL groups CO3-610C levels increased significantly compared to sham groups (mean values: 2 weeks, post-surgery sham 39.7 ng/ml, BDL 100.3 ng/ml; average increase between the groups was 153%; 4 weeks post-surgery, sham 39.7, BDL 92.6 ng/ml, average increase between the groups was 133%) (FIG. 5 panels A and B). There were no changes in the sham groups. CTX-II levels indicating collagen type II degradation did not change in the sham or BDL groups (FIG. 5 panel C).

Type III Collagen Gene Expression:

FIG. 6 shows Type III collagen gene expression in BDL or sham-operated rats. Data are shown as mean+standard of mean; 2 weeks post-surgery P<0.0001 and 4 weeks post-surgery P=0.0006

Type III collagen a1 chain mRNA increased significantly in both BDL groups compared with sham-operated rats.

Western Blot and Densitometry:

FIG. 7 shows changes in the expression of CO3-610C in the liver of rats in BDL- and sham-operated groups assessed by A) Western blot 2 and 4 weeks post-surgery and B) Bands from western blot quantified by densitometry.

Western blot analysis showed very low levels of CO3-610C in sham-operated rats (FIG. 7 panel A). At and after 2 weeks post-surgery CO3-610C levels prominently increased (FIG. 7 panel A). Results were quantified by densitometry analysis (FIG. 7 panel B).

Histology Image Analysis:

FIG. 8 panel A shows in the top row histology sections from BDL- or sham-operated rats stained with Sirius Red. The bottom row shows masked histology sections for quantifying total collagen content (red colour) in the liver. Panel B shows total collagen quantified by Visiopharm software—2 weeks post-surgery P=0.0081; 4 weeks post-surgery P=0.0047

Histology sections stained with Sirius Red and enhanced using Visiopharm software showed increasing collagen content over time in BDL-operated rats. (FIG. 8 panel A). The red color in the mask representing collagen was quantified using the same software (FIG. 8 panel B) and confirmed a significant increase in total collagen content in BDL-operated rats compared with sham-operated rats (2 weeks post-surgery P=0.0081; 4 weeks post-surgery P=0.0047).

Correlation:

FIG. 9 panel A shows a correlation of Col3a1 to CO3-610C was found with R²=0.6993, P<0.0001. In panel B, a correlation of CO3-610C to % collagen was found with R²=0.2278 and P=0.0050. In panel C a correlation of Col3a1 to % collagen was found with R²=0.5409, P<0.0001.

Correlations were found of the following: Col3a1 mRNA to CO3-610C with R²=0.6993 and P<0.0001 (FIG. 9A), and CO3-610C to % collagen quantified by visiopharm with R²=0.2278 and P=0.0050 (FIG. 9B), and Col3a1 mRNA to % collagen quantified by visiopharm with R²=0.5409 and P<0.0001 (FIG. 9C).

ECM remodelling is an integrated process of tissue development, maintenance and pathogenesis. Proteolytic activity is essential in this process for cell migration, removal of damaged tissue, and sequestering of new proteins, for the correct and optimal tissue orientation and quality (108:109). The specific matrix degradation products, neo-epitopes, may be important for the identification of new biochemical markers of liver fibrosis matrix turnover and understanding fibrosis pathogenesis. At present there are no available measuring techniques, nor biochemical markers, that allow for assessment of ECM remodeling in the pathogenesis of fibrosis.

In this example, to investigate the CO3-610C marker under in vivo situations, 6 months BDL rats were chosen, as they previously have been shown to have a lower collagen remodelling compared to younger rats. The rats are skeletally mature, and the growth plate is almost dormant, thereby contributing to a much lower extent to the overall collagen turnover. This influences the sensitivity and specificity for biomarker. These rats clearly presented with hepatic fibrosis, as evaluated by both quantitative histological analysis, and enlargement with increased weight, thus the model was an appropriate one to look for evidence of ECM remodeling, in particular for evidence of collagen type III in serum.

The present data clearly demonstrate the neo-epitope CO3-610C from MMP-9 mediated collagen type III degradation is a diagnostic biochemical marker for liver fibrosis with an average increases in serum of up to 153% from sham to BDL-operated rats.

To further investigate the biological rationale for the increased CO3-610C marker, we did protein extractions from healthy and diseased livers. By western blotting, we identified a predominant band, suggesting this to be an abundant protein fragment in diseased but not healthy livers. This provides evidence for the pathological accuracy of this novel marker.

To further investigate the pathological turnover representation of the liver, we measured type III collagen mRNA. We found an increase of mRNA in the BDL rats compared to those undergoing the sham operation, which correlates with previous findings. These data strongly suggest that liver fibrosis is not only an accumulation of ECM proteins, but also an accelerated turnover situation, in which both tissue formation and tissue degradation both are highly up regulated. Tissue formation outstrips tissue degradation, leading to an accumulation of scar tissue over time. Previous investigators have used other matrix turnover proteins to assess liver fibrosis, one being the type III collagen formation marker N-terminal type III pro-collagen. This marker represents collagen type III formation and has shown to be increased in liver fibrosis in previous studies.

To further understand and the dynamics of the biochemical makers CO3-610C, we did a range of correlations. Most importantly, there was a significant correlation of CO3-610C to the extent of fibrosis measured in the liver by quantitative histology. The level of liver fibrosis was correlated to the expression levels of the mRNA of collagen type III. Finally, the CO3-610C correlated to mRNA of collagen type III in the liver. Taken together, there was a significant correlation of the pathological processes in the liver with the levels of the systemic biochemical markers CO3-610C. In addition the tissue extractions provided evidence that the circulation levels were locally produced.

Example 6 ELISA on Human Serum Samples

Human serum samples were obtained from patients with Chronic Obstructive Pulmonary Disease (COPD) (n=5), scleroderma (n=5), chronic hepatitis C virus infection (n=5), and healthy controls (n=5). The serum samples were tested in the CO3-610 ELISA (see Example 4 above) to determine the concentration of CO3-610 fragments. Results are shown in FIG. 10. While serum samples from the healthy subjects had concentration of CO3-610 fragments below 30 ng/ml, the diseased subjects were found to have elevated levels in circulation suggesting massive tissue remodelling in the affected fibrotic tissues.

Example 7 Reactivity of Clone Nb94

Mice were immunized with synthetic peptide KAFVFP (SEQ ID NO1167) conjugated to ovalbumin (KAFVFPKESD-GGC-OVA (SEQ ID NO1049)), spleen cells were used for fusion, and monoclonal antibodies tested for reactivity to biotinylated KAFVFP (SEQ ID NO 1167), i.e. (KAFVFPKESD-biotin(SEQ ID NO1049)) immobilized in wells of microtitre plates precoated with streptavidin. Antibodies binding to biotinylated KAFVFPKESD(SEQ ID NO1049), which could be inhibited by co-incubation with KAFVFPKESD (SEQ ID NO1049) but not the elongated peptide RKAFVFPKESD (SEQ ID NO1166), were selected for further characterization. The preferred monoclonal antibody was designated NB94-37-1A7. Using a competition ELISA, essentially as described above with biotinylated KAFVFPKESD (SEQ ID NO1049) (used at 0.15 ng/ml) immobilized in the wells of streptavidin-coated microtitre plates, an incubation step (90 minutes at 20° C.) with sample and monoclonal antibody NB94-37-1A7 followed by a washing step, and then addition of peroxidase-conjugated anti-mouse immunoglobulins. For competition the following material was used in 2-fold dilutions; (1) the synthetic KAFVFP (SEQ ID NO1167) peptide; (2) a nonsense peptide (KNEGTG) unrelated to CRP; (3) a pool of human serum samples; (4) CRP proteolytically cleaved with MMP3 for 7 days, subsequently stopped by addition of EDTA to block protease activity, and stored at −80° C. until testing; (5) same as (4) but using MMP8 instead of MMP3; (6) same as (4) except using Cathepsin K (for 2 days) instead of MMP3 (and E64 as inhibitor to block Cathepsin K activity).

The data demonstrate that monoclonal antibody NB94-37-1A7 binds strongly to the synthetic peptide KAFVFPKESD (SEQ ID NO1049), and with CPR cleaved with MMP3 and MMP8. Cleavage of CRP with Cathepsin K release less analyte recognized by monoclonal antibody NB94-37-1A7. Finally, the data shows that the antibody binds to peptide fragments in human serum confirming the presence of this sequence in circulating peptide fragments.

Example 8 CO3 in Biological Relevant Samples: CO3 Levels in Carbon Tetrachloride (CC14)-Induced Cirrhosis in Rats

Animals and Induction of Cirrhosis:

This study included 52 male Wistar rats with fibrosis or cirrhosis and 35 male Wistar control rats. To cause them to develop fibrosis or cirrhosis three-month old animals were included in an induction program with carbon tetrachloride (CC14) and Phenobarbital treatment. CCl₄ was administered by inhalation twice weekly and phenobarbital (0.3 g/l) added to the drinking water. Animals were allowed free access to water and food throughout the study.

Fibrosis Quantification:

Liver sections (4 μm) were stained in 0.1% Sirius red F3B (Sigma-Aldrich, St. Louis, Mo.) in saturated picric acid (Sigma-Aldrich). Relative fibrosis area (expressed as a percentage of total liver area) was assessed by analyzing 36 fields of Sirius red-stained liver sections per animal. Each field was acquired at 10× magnification [E600 microscope (Nikon) and RT-Slider SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, Mich.). Results were analyzed using a computerized Bioquant Life Science morphometry system. To evaluate the relative fibrosis area, the measured collagen area was divided by the net field area and then multiplied by 100. Subtraction of vascular luminal area from the total field area yielded the final calculation of the net fibrosis area. From each animal analyzed, the amount of fibrosis as percentage was measured and the average value presented.

Classification of Groups According to their Fibrosis/Cirrhosis Stage:

Animals were classified into 4 different stages of fibrosis and cirrhosis (Group A: moderate fibrosis, group B: advanced fibrosis, Group C: moderate cirrhosis, and Group D: advanced cirrhosis) that were determined by the percentage of Sirius red positive liver area (Group A: <5%, Group B: 5 to 10%, Group C: 10 to 15% and Group D: >15%). For this purpose, control and fibrotic/cirrhotic rats were studied considering four different time points during the CC14 treatment: 8, 12, 16 and 20 weeks after starting the cirrhosis induction program.

Hyaluronic Acid Measurement:

Serum hyaluronan was measured using a sandwich ELISA kit (R&D Systems Inc., Minneapolis, Minn., USA).

Statistics:

Statistical analysis of results was performed by unpaired Student's t tests when appropriate. Data were expressed as mean±S.E.M., and they were considered significant at a p level of 0.05 or less.

Study Design:

Animals included in this protocol were randomly assigned to one of the following groups: A/eight weeks of CCl₄ treatment, B/twelve weeks of CCl₄ treatment, C/sixteen weeks of CCl₄ treatment and D/twenty weeks of CCl₄ treatment. In parallel, four control groups were studied at the same time points. Thirteen fibrotic rats and seven control rats were included in each group. At the end of the study, rats were placed in standard metabolic cages (Tecniplast Deutschland, Hohenpeissenberg, Germany) during an adaptation period of 3 days before proceeding with the twenty-four-hour urine collection. Urinary volumes were determined gravimetrically. During the adaptation period, rats were allowed to get free access to tap water and food. Then, 24-hour urine samples were centrifuged for 5 min at 2,500 rpm and aliquoted into ten polypropylene tubes (400 μL each). Urine samples were stored at −80° C. for subsequent analysis.

At scheduled necropsies, rats were weighed, anesthetized with pentobarbital (50 mg/kl) and decapitated. Blood were collected and allowed to stand at room temperature for 20 min to allow clotting and then centrifuged for 10 min at 2500 rpm. Serum were collected in polypropylene tubes aliquots (400 μl each) and transferred via dry ice to a −80° C. freezer. Collection of baseline blood samples at the beginning of the CCl₄ treatment was not considered in order to avoid additional intervention that may increase the risk of infection and/or introduce modifications in the experimental model that may compromise the evolution of the induced pathophysiological process. For histology and Sirius red staining, half of the left lobe of the liver were placed in 10% neutral buffered formalin for 16 hours, embedded in paraffin and sectioned into 4-μm-thick slices. After liver fibrosis quantification, the unused paraffin block material was preserved for biomarker quantification. The other half of the left lobe was flash-frozen in liquid nitrogen and stored for Western blot, RT-PCR or immunohistochemical analysis. Measurements of liver fibrotic area, serum and urine osmolality, Na⁺ and K⁺, albumin, creatinine, alanine amino-transferase and lactate dehydrogenase were made according to the Material and Methods section.

Results:

Histological Validation of the Model:

Liver collagen was quantified in all study animals by Sirius red staining of liver slices. The final data for each animal was taken as the average of red staining observed in 36 consecutive microscope fields (FIG. 12).

FIG. 12 shows representative pictures from two sets of 36 images used to quantify collagen accumulation in liver in rat #1 (left) and rat #43 (right) treated with carbon tetrachloride for eight and twenty weeks respectively.

The serum CO3 marker shows statistically significant increases in both fibrotic and cirrhotic rats compared to control rats. Animals were classified according to a fully automated syrius red staining of the liver procedure used to quantify fibrosis (FIGS. 13 and 14).

FIG. 13 shows serum CO3 levels in CCl₄ inhalation and control rats as performed in Hospital Clinic (Barcelona). Each point represents one animal. Rats were classified according a computerized image analysis method of syrius red staining of the liver used to quantify fibrosis.

When quantitative values of serum CO3 and syrius red staining of the liver were studied in each individual animal, we found a statistically significant correlation between the two variables (R2=0.4087; n=21) (FIG. 14).

We have compared the levels of CO3-610C with the serological benchmark of liver fibrosis hyaluronic acid (HA). HA levels were quantified with a commercial ELISA kit and results show significant elevations of this ECM component in cirrhotic rats vs. fibrotic animals (FIGS. 15 and 16).

The correlation of CO3 to Sirius red outperformed that of HA. More than seventy percent of the variation in liver fibrosis histological quantification can be explained by the serological measurement of CO3. The remaining thirty percent is due to unknown variables or inherent variability. Instead only 25% of liver fibrosis can be explained by measuring hyaluronic acid (FIG. 15).

As expected from the previous result no correlation could be found between CO3 and hyaluronic acid suggesting that they are the result of two independent pathophysiological processes in the development of liver fibrosis (FIG. 17).

Example 9 Bleomycin Induced Skin Fibrosis in Mice

Mice were treated by application to the skin of PBS or bleomycin. Increasing levels in urine of the MMP-9 mediated collagen III (CO3) degradation fragment CO3-610 were associated with skin fibrosis progression in mice.

FIG. 18 shows a skin section from a PBS treated mouse at 8 weeks of treatment (panel A) and a skin section from Bleomycin treated mouse at 8 weeks of treatment (panel B). Skin thickness increase between PBS (n=7/time point) and Bleomycin (n=13/time point) treated mice for 2 weeks (P=0.0029), 4 weeks (P=0.0004), 6 weeks (P<0.0001) and 8 weeks (P<0.0001) is plotted in panels C and D. Overall skin thickness increase between PBS (n=28) and Bleomycin (n=52) treated mice for the duration of the study (P<0.0001). Skin width was calculated by Visiopharm software as an overall number per skin section instead of sampling pictures.

FIG. 19 shows CO3-610 urine assay results which demonstrate a significant increase throughout the time points of the study. The figure shows result per time point (n=7 PBS, n=13 Bleomycin treated per termination point) and collective CO3-610 levels for all time points (n=28 PBS and n=52 Bleomycin treated mice). 2 weeks P=0.0008, 4 weeks P<0.0001, 6 weeks P<0.0001, 8 weeks P<0.0001 and overall P<0.0001.

FIG. 20 shows a CO3-610 Western blots image with control C and Bleomycin B after 2 and 8 weeks treatment (panel A). CO3-610 densitometry measurements for all time points (n=7 PBS and n=13 Bleomycin treated per termination point) and collective CO3-610 levels (n=28 PBS and n=52 Bleomycin treated mice) are shown in panel B, demonstrating a statistically significant increase of CO3-610 levels (P<0.0001).

As seen in FIG. 21, CO3-610 levels in urine assay were found to be correlated with skin thickness progression, and therefore total collagen deposition r=0.4883, R2=0.2384.

As seen in FIG. 22, statistically significant correlation was found (r=0.6528, P<0.0001) between results from the CO3-610 ELISA urine assay and Western blot densitometry measurements.

In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used in the sense of ‘including’ rather than in to mean ‘consisting of’. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

REFERENCE LIST

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention and the following claims.

INCORPORATION BY REFERENCE

All publications, patent applications and patents identified herein are expressly incorporated herein by reference in their entirety. 

The invention claimed is:
 1. A method of diagnosis or of quantitation of fibrosis comprising obtaining a patient biofluid sample, conducting an immunoassay to measure fragments of collagen type VI having an N- or C-terminal neo epitope formed by cleavage of collagen type VI by MMP2, MMP9, MMP13, FAP-1 or FAP-2, said fragments being naturally present in said sample, and associating an elevation of said measure in said patient above a normal level with the presence or extent of fibrosis, wherein said immunoassay is conducted by a method comprising: contacting the fragments of collagen type VI having said N- or C-terminal neo epitope that are naturally present in said sample with an immunological binding partner specifically reactive with the N- or C-terminal neo-epitope but not reactive with intact collagen type VI, and measuring the extent of binding of the N- or C-terminal neo-epitope to said immunological binding partner to measure therein fragments comprising said neo-epitope, wherein said immunological binding partner is raised against a synthetic peptide corresponding to an N-terminal neo epitope amino acid sequence formed by cleavage of collagen type VI by MMP2, MMP9, MMP13, FAP-1 or FAP-2 and specifically binds a neo-epitope constituted by said N-terminal amino acid sequence, said N-terminal amino acid sequence selected from the group consisting of: Collagen type VI YRGPEG SEQ ID NO PIGPKG SEQ ID NO GIGIGN SEQ ID NO 883 865 885 ISGPRG SEQ ID NO PGPAGP SEQ ID NO VAAKPA SEQ ID NO 886 887 888 GEPGPP SEQ ID NO RGPIGS SEQ ID NO PPPPQP SEQ ID NO 675 889 890 AQGPAG SEQ ID NO LIGEQG SEQ ID NO PGLIGE SEQ ID NO 891 892 893 GEPGLN SEQ ID NO IGPKGI SEQ ID NO VAVVQH SEQ ID NO 894 895 896 FGPSAA SEQ ID NO GPKGET SEQ ID NO LGPMGV SEQ ID NO 897 898 899 PGEPGP SEQ ID NO 784

or said immunological binding partner is raised against a synthetic peptide corresponding to a C-terminal neo epitope amino acid sequence formed by cleavage of collagen type VI by MMP2, MMP9, MMP13, FAP-1 or FAP-2 and specifically binds a neo-epitope constituted by said C-terminal amino acid sequence, said C-terminal amino acid sequence selected from the group consisting of: Collagen type VI GDEGPP SEQ ID NO GNADIT SEQ ID NO PAGPPG SEQ ID NO 900 901 133 DPGLMG SEQ ID NO PEVPRP SEQ ID NO TGPKGI SEQ ID NO 902 903 904 GDEGGP SEQ ID NO PARSAS SEQ ID NO TPAPPG SEQ ID NO 905 906 915 ISGPRG SEQ ID NO GISGPR SEQ ID NO GIGNRG SEQ ID NO 886 907 908 YRGYPG SEQ ID NO KVEFSL SEQ ID NO GVPGRD SEQ ID NO 909 910 911 PGETGK SEQ ID NO RTGPLG SEQ ID NO APGERG SEQ ID NO 912 913
 914.


2. A method of immunoassay to measure fragments of collagen type VI having an N- or C-terminal neo epitope formed by cleavage of collagen type VI by MMP2, MMP9, MMP13, FAP-1 or FAP-2, said fragments being naturally present in a biofluid sample, wherein said immunoassay is conducted by a method comprising: contacting the fragments of collagen type VI having said N- or C-terminal neo epitope that are naturally present in said sample with an immunological binding partner specifically reactive with the N- or C-terminal neo-epitope but not reactive with intact collagen type VI, and measuring the extent of binding of the N- or C-terminal neo-epitope to said immunological binding partner to measure therein fragments comprising said neo-epitope, wherein said immunological binding partner is raised against a synthetic peptide corresponding to an N-terminal neo epitope amino acid sequence formed by cleavage of collagen type VI by MMP2, MMP9, MMP13, FAP-1 or FAP-2 and specifically binds a neo-epitope constituted by said N-terminal amino acid sequence, said N-terminal amino acid sequence selected from the group consisting of: Collagen type VI YRGPEG SEQ ID NO PIGPKG SEQ ID NO GIGIGN SEQ ID NO 883 865 885 ISGPRG SEQ ID NO PGPAGP SEQ ID NO VAAKPA SEQ ID NO 886 887 888 GEPGPP SEQ ID NO RGPIGS SEQ ID NO PPPPQP SEQ ID NO 675 889 890 AQGPAG SEQ ID NO LIGEQG SEQ ID NO PGLIGE SEQ ID NO 891 892 893 GEPGLN SEQ ID NO IGPKGI SEQ ID NO VAVVQH SEQ ID NO 894 895 896 FGPSAA SEQ ID NO GPKGET SEQ ID NO LGPMGV SEQ ID NO 897 898 899 PGEPGP SEQ ID NO 784

or wherein said immunological binding partner is raised against a synthetic peptide corresponding to a C-terminal neo epitope amino acid sequence formed by cleavage of collagen type VI by MMP2, MMP9, MMP13, FAP-1 or FAP-2 and specifically binds a neo-epitope constituted by said C-terminal amino acid sequence, said C-terminal amino acid sequence selected from the group consisting of: Collagen type VI GDEGPP SEQ ID NO GNADIT SEQ ID NO PAGPPG SEQ ID NO 900 901 133 DPGLMG SEQ ID NO PEVPRP SEQ ID NO TGPKGI SEQ ID NO 902 903 904 GDEGGP SEQ ID NO PARSAS SEQ ID NO TPAPPG SEQ ID NO 905 906 915 ISGPRG SEQ ID NO GISGPR SEQ ID NO GIGNRG SEQ ID NO 886 907 908 YRGYPG SEQ ID NO KVEFSL SEQ ID NO GVPGRD SEQ ID NO 909 910 911 PGETGK SEQ ID NO RTGPLG SEQ ID NO APGERG SEQ ID NO 912 913
 914.


3. A method of immunoassay to measure fragments of collagen type VI having an N-terminal neo epitope formed by cleavage of collagen type VI by MMP2, said fragments being naturally present in a biofluid sample, wherein said immunoassay is conducted by a method comprising: contacting the fragments of collagen type VI having said N-terminal neo epitope that are naturally present in said sample with an immunological binding partner specifically reactive with the N-terminal neo-epitope but not reactive with intact collagen type VI, and measuring the extent of binding of the N-terminal neo-epitope to said immunological binding partner to measure therein fragments comprising said neo-epitope, wherein said immunological binding partner is raised against a synthetic peptide corresponding to an N-terminal neo epitope amino acid sequence formed by cleavage of collagen type VI by MMP2 and specifically binds a neo-epitope constituted by said N-terminal neo epitope amino acid sequence, said N-terminal amino acid sequence being YRGPEG SEQ ID NO883. 